All thymocyte subpopulations derive from intrathymic precursors which are double negative (DN) for Lyt-2 and L3T4 differentiation antigens. Although nearly half of DN cells express a receptor for interleukin 2 (IL 2R), they respond poorly to IL 2. DN cell proliferation can be obtained in the presence of various exogenous stimuli, but the in vivo signal for DN cell response to IL 2 remains unclear. We show in the present report that phagocytic cells of the thymic reticulum are able to induce the proliferation of DN thymocytes in the presence of recombinant IL 2 (rIL 2). Cell-to-cell contact is needed for this effect. Antibodies directed against class I MHC antigens but not against class II can inhibit DN cell proliferation. DNA-synthetizing cells were labeled by incubation with 10 microM bromodeoxyuridine either before or at various times during the culture period. Bromodeoxyuridine was then detected in the DNA of proliferating cells and/or their progeny already stained with anti-Lyt-2 and L3T4 antibodies. During the initial 16 h and independently of culture conditions, 16-25% of the cells expressed surface antigens and 50-65% of them derived from DN cells which were in S phase just before culture; these differentiated cells had a very short life span. In the second culture period, the presence of both rIL 2 and thymic accessory cells was necessary for cell survival. In these conditions, DN cell number and proliferation rate were constant and a low number of Lyt-2+ and/or L3T4+ cells was continuously generated. Thymic accessory cells therefore appear to provide the signal(s) necessary for IL 2-induced proliferation of thymocyte precursors. Implications of these findings for normal in vivo intrathymic proliferation and differentiation are discussed.