The mechanisms by which growth factors stimulate metabolism and cell proliferation are largely unknown. Recent evidence suggests that mitogens rapidly activate a Na+/H+ exchange mechanism in the plasma membrane of their target cells, implicating cytoplasmic pH (pH1) as a potential 'messenger'. Indeed, growth stimulation of quiescent fibroblasts leads to intracellular alkalinization at approximately 1 h after mitogen addition, as measured by weak-acid distribution methods. We have used an internalized fluorescent pH1 indicator to examine the pH1-regulating mechanisms in diploid human fibroblasts and to obtain the first continuous pH1 recordings of the response to growth factors. We report here that (1) pH1 in human fibroblasts is controlled by a membrane-bound Na+/H+ exchanger, which rapidly restores pH1 after an acute cytoplasmic acidification, and (2) epidermal growth factor (EGF) and serum factors induce a rapid and persistent elevation of pH1 by modifying the pH1 sensitivity of the Na+H+ exchanger. We conclude that in addition to having a basic role in pH1 regulation, Na+/H+ exchange may function as a transmembrane signal transducer in the action of growth factors.