Observations from our own laboratories, as well as those of others, have demonstrated the critical role of histocompatibility gene products in governing the cell-cell interactions concerned with development and regulation of immune responses in several species (8-12). In mice, the relevant genes concerned have been shown to be located in the K end of the H-2 complex, i.e. in the K and/or I See PDF for Structure regions (13, 14). These discoveries have placed histocompatibility gene products on a more complex level of biologic function than was heretofore generally considered (15). Thus, the hypothesis was made from these observations that genes in the H-2 complex coded for products involved in the development of effective cell-cell interactions in the immune response (8, 9, 15). The recent identification of cell surface macromolecules on lymphocytes and macrophages, that may be distinct from immune response gene products but are likewise coded for by genes in the I region, has provided a group of suitable candidate molecules for such a role (2). In our initial studies on the biological and biochemical characteristics of AEF, we were impressed by the apparent preferential activity of the highly purified AEF preparations on B lymphocytes syngeneic to the activated T-cell population from which the AEF was obtained (1). Since a prediction of the aforementioned hypothesis is, of course, that the active molecules involved in regulatory immunocompetent cell interactions are gene products of the H-2 complex, and, accordingly, should be reactive with antisera directed against components of this complex, we were prompted to perform the appropriate analyses on our preparation of AEF. The experiments presented here demonstrate that the enhancing activity of AEF obtained from T cells of the H-2(d) haplotype can be specifically removed by immunoadsorbents prepared from antisera reactive with la molecules of the H-2(d) allele. Identical results were obtained in experiments with both direct and indirect absorption procedures. The possibility that the reaction of AEF with the B10.A anti-B10 (anti-Ia.8) antiserum resulted in release of some components that were in turn toxic to the cultured cells, has been made unlikely in these studies by the use of a direct adsorption method utilizing an immunoadsorbent prepared from thoroughly washed glutaraldehyde-linked antibodies. The results obtained with the (B6A)F(1) anti-B10.D2 antiserum deserve some comment. This antiserum contains antibodies directed predominantly against the H-2K region specificity, H-2.31, but may also be reactive with recently determined Ia(d) specificities (5). The capacity of this antiserum to directly absorb approximately 45% of the AEF activity at the lowest concentration of AEF employed (Fig. 1) could be interpreted to indicate the reactivity of AEF with anti-H-2K antibodies. However, the data presented here are also consistent with the interpretation that partial adsorption by the direct immunoadsorbent and lack of adsorption by the indirect method (in which only a high concentration of AEF was incubated with the alloantisera) reflect reactivity of AEF with anti-Ia(d) antibodies present in this antiserum. We conclude, therefore, that the biologically active enhancing moieties of AEF bear Ia determinants and therefore are most probably gene products of the I region of the H-2 gene complex. Recent data from other investigators have shown that an antigen-specific T-cell product could be specifically adsorbed by immunoadsorbents prepared from antisera directed against the K end of H-2 (16). Since the latter antisera may contain antibodies reactive with specificities of both K and I regions, it is possible that the use of selective anti-Ia sera may yield results consistent with those presented here. Taken collectively, these observations indicate that I-region gene products may be intimately involved in the mechanism of cell-cell interactions and responsible for the regulation of immune responses.