Addition of platelet-derived growth factor, epidermal growth factor, or serum to quiescent human fibroblasts, loaded with the fluorescent Ca2+ indicator quin-2, causes an immediate, up to 3-fold, rise in cytoplasmic free Ca2+ concentration [( Ca2+]i). In contrast, insulin and tumor-promoting phorbol ester have no effect on [Ca2+]i. The mitogen-induced [Ca2+]i response is initiated within a few s, reaches a maximum by 20-40 s, and then slowly declines to a new steady level. The [Ca2+]i response is not prevented by removal of external Ca2+ and is independent of the transmembrane Na+ gradient and membrane potential. It is concluded that platelet-derived growth factor, epidermal growth factor, and serum rapidly mobilize Ca2+ from intracellular stores, presumably due to the prior breakdown of inositol phospholipids, and that the resulting rise in [Ca2+]i may function as an initial signal in growth factor action.