Activation of naive CD4+ T cells is essential for the induction of primary immune responses. However, this subset is less responsive to signaling via T cell receptor/CD3 (TcR/CD3) complex than memory CD4+ cells. For mitogenic activation of T cells, in addition to triggering of the TcR/CD3 complex, costimulatory signals are required that can be generated by surface structures present on the antigen-presenting cells. We investigated here whether differences in responsiveness to TcR/CD3 stimulation of naive and memory cells can be overcome by the costimulatory pathway B7/CD28. Using a B7-dependent system we show that even in the presence of optimal CD28 costimulation, CD4+ naive cells still have more stringent TcR/CD3 activation requirements than memory cells. Furthermore, titration of the B7 signal revealed that for activation of naive CD4+ cells a higher level of cross-linking of CD28 molecules is required than for memory cells. Thus, our results show that at least two signals are required for activation of both CD4+ memory and naive cells, but that for activation of naive cells higher cross-linking of both CD3 and CD28 molecules is necessary.