In response to growth factors, quiescent fibroblast mutants lacking Na+/H+ exchange activity fail to elevate their cytoplasmic pH (pHi) and to reinitiate DNA synthesis at neutral and acidic pHo. A pHi threshold of approximately 7.2 exists, below which growth factors cannot set in motion the Go to S phase transition. Restoration of the pHi defect in mutant cells restores the wild-type phenotype. These findings, combined with the properties of another class of mutants able to grow at very low pHo, demonstrate that pHi, modulated by growth factor activation of the Na+/H+ antiporter, plays a determinant role in growth control.