Primary CML was generated in strain combinations 4R anti-2R, R107 anti-3R, 7R anti-9R, and GD anti-R101-combinations differing only in the chromosomal interval between the I-A subregion and the Ss locus. No CML could be obtained in any of the reciprocal combinations of these strains. This unidirectionality of the CML reaction correlates with the expression or nonexpression of the E molecules encoded by this interval: the reaction occurred in combinations in which the responder strain lacked and the stimulator strain expressed the E molecules in the cell membrane. The CML reaction was positive when tested on LPS-stimulated blast cells but weak on Con A-stimulated blasts and negative on Ia-negative tumor cells. The reaction could partially be inhibited by monoclonal antibodies to the Ia.m7 determinant presumably carried gy the E alpha chain; it was not inhibited by monoclonal antibodies specific for Ia determinants carried by the A molecule. Cytotoxic lymphocytes specific for a particular combination of E beta and E alpha chains reacted with all cells expressing the particular E beta chain, no matter what the origin of the E alpha chain associated with the E beta chain was. Attempts to generate cytotoxic lymphocytes specifically reactive with allotypic determinants on E alpha chains failed. In F1 hybrids expressing one type of E alpha chain and two types of E beta chain, the single E alpha chain was found to associate with both beta chains, producing two types of E molecule. We conclude from these experiments that the CML determinants detected in the strain combinations used are encoded by the same loci as those coding for the serologically detectable Ia determinants. The CML determinants are carried by the E beta chains; the E alpha chain does not contribute in any way to the specificity of determinant recognition by the cytotoxic lymphocytes. No evidence for allotypic variation of the E alpha chain as detected by the CML assay could be found in this study.