Mitogen-induced intracellular alkalinization mediated by activation of a Na+/H+ antiporter is a common feature of eukaryotic cells stimulated to divide. A Chinese hamster fibroblast mutant (PS120) lacking Na+/H+ antiport activity (Pouysségur et al., Proc natl acad sci US 81 (1984) 4833) [42] possesses an intracellular pH (pHi) 0.2-0.3 units lower than the wild type (CCL39) and requires a more alkaline pHout (pHo) for growth. Here, we show that serum-stimulated ribosomal protein S6 phosphorylation, protein synthesis activation and DNA synthesis re-initiation are pH-regulated events that display a similar threshold pHo value (6.60) in CCL39 cells. pH-Dependencies for initiation of all three events are shifted toward higher pHo values in the mutant PS120, indicating that growth factor-induced alkalinization has a permissive effect on the pleiotypic response. However, cytoplasmic alkalinization per se is insufficient to trigger S6 phosphorylation, polysome formation, and subsequent DNA synthesis. Transient exposure to a non-permissive pHo (6.5) inhibits both the rate of leucine incorporation into proteins and the progression through the G1 phase of the cell cycle. In contrast, cells committed to DNA synthesis are unaltered by the acidic pHo. These observations suggest that pHi by controlling the rate of protein synthesis play a determinant role in the control of cell division.