Antigen-induced T cell activation can be separated into early recognitive events and later proliferative events. After in vitro challenge of primed lymphocytes with antigen, enhanced transmembrane transport of α-aminoisobutyric acid (14C-AIB), a nonutilizable amino acid, can be demonstrated within 4 to 8 hr, and is at or near maximum by 8 to 16 hr of culture. The response is antigen specific and requires that the primed animal is a genetic responder to the immunizing antigen. Therefore, AIB uptake was utilized as a probe of antigen recognition and compared to tritiated thymidine incorporation, a measure of DNA synthesis. Colchicine, cytochalasin B, and Ca++ depletion have been reported to block DNA synthesis in activated lymphocytes and to interfere with macrophage-T cell interaction. We confirmed that these agents effectively abrogated antigen-induced DNA synthesis under conditions which, in contrast, had little effect on antigen-induced AIB uptake. We interpret these results to indicate at least a two-signal model for antigen-induced T cell proliferation. An initial signal is generated by the interaction of antigen with its membrane receptor. Lymphocyte activation commences but at some step before the initiation of S-phase, a second critical event, which can be blocked by colchicine, cytochalasin B, and Ca++ depletion, must occur. Since those agents block macrophage-T cell binding, the second signal may be the result of that cell-cell binding.