We investigated the effects of rapamycin (RAP) on cell cycle progression and protein synthesis in mitogen-activated primary T lymphocytes. Stimulation of resting human T lymphocytes with phorbol ester and calcium ionophore rendered cells capable of initiating DNA synthesis within 30 h; roughly 60% of the cells entered the first G2/M phase of the cell cycle within 96 h. Addition of RAP delayed the entry into S phase by 9 h, although a similar percentage (approximately 50%) of cells entered the first G2/M phase and proliferated. On this basis, we concluded that RAP primarily induced a G1 prolongation without blocking cell cycle progression. Addition of the co-mitogens to resting T lymphocytes up-regulated the translation of ribosomal protein mRNA concurrent with activation of p70s6k. RAP inhibited this translational up-regulation of ribosomal protein mRNA as well as the activation of p70s6k without affecting translation of nonribosomal protein mRNA. RAP also prevented the synthesis and accumulation of ribosomal proteins. Further, this failure to increase ribosomal proteins, which probably reflects the failure to increase numbers of ribosomes, resulted in suppression of the synthesis of total cellular protein and a delay in the escalation of cell size. RAP-treated cells eventually initiated DNA synthesis when cell size became equivalent to that of the control cells entering S phase of the cell cycle. Thus, inhibition of protein synthesis caused by the primary inhibition of ribosomal protein mRNA translation probably explains the effect of RAP on cell cycle progression of mitogen-activated resting T lymphocytes.