A fetal thymus organ culture system has been developed to study the differentiation of murine thymus-derived immunocompetent cells (T cells) such that cell yields can be easily monitored. This system has been used to study the effects of monoclonal anti-I-A antibodies on the growth of T cells. The addition of anti-I-A antibodies, but not anti-H2K monoclonal antibodies, to fetal thymus organ cultures resulted in a decreased yield of lymphoid cells. Anti-I-A-treated cultures did not produce cells that gave an immune response in MLC assays. Anti-I-A antibodies stained a small subpopulation of nonlymphoid cells in untreated cultures by indirect immunofluorescence that were no longer detectable in cultures that had been pretreated with anti-I-A antibody. Culture of fetal thymus lobes at low temperature (20 degrees C) for 1 wk resulted in a decrease in lymphocyte production, as well as a concomitant increase in the frequency of Ia-positive nonlymphoid cells. Co-culture of fetal liver or anti-thy-1 plus complement-treated adult bone marrow with such Ia-positive cell-enriched fetal thymus lobes at 37 degrees C resulted in the production of T cells. Anti-Thy-1.1 or -1.2 staining by indirect immunofluorescence of cells obtained from co-cultures that differed at the Thy-1 locus showed that the T cells produced were derived from the bone marrow or fetal liver. T cell production occurred in both syngeneic and allogeneic cocultures. However, if co-cultures were made by using 14-day gestation fetal thymus instead of fetal liver or bone marrow as donors of T cell precursors, T cell growth was observed only in syngeneic combinations. These results suggest that Ia-positive nonlymphoid cells play a role in the development of T cells in the fetal thymus, and that "thymus processed" T cell progenitors (but not the more immature progenitors in the fetal liver or bone marrow) are self-Ia restricted in their differentiation.