Receptor-activated breakdown of complex sphingolipids has been proposed as a mechanism for generating sphingoid base-containing putative second messenger molecules whose actions may modulate responses to extracellular signals. In human epidermoid carcinoma A431 cells, sphingosine (1-10 microM) by itself had no effect on intracellular free calcium concentrations ([Ca2+]i), yet within seconds, markedly enhanced the epidermal growth factor (EGF)-evoked Ca2+ influx (by up to 2-fold), but failed to alter Ca2+ release from the intracellular stores. Ca2+ signals evoked by serum were not affected by sphingosine. The response to sphingosine was dose-dependent and saturable, exhibiting an EC50 of 2.3 microM. In contrast, a ceramide, N-acetylsphingosine (10 microM), sphingosine 1-phosphate (10 microM), and sphingosylphosphorylcholine (10 microM) inhibited EGF-evoked elevations in [Ca2+]i. The latter two compounds by themselves transiently increased [Ca2+]i. N-Octanoylsphingosine, N,N-dimethylsphingosine, sphingomyelin, and stearylamine were inactive. The potentiation of calcium signals by sphingosine occurred at all concentrations of EGF tested (0.15-15 nM) and did not alter the EGF receptor protein kinase activity as determined by antiphosphotyrosine immunoblotting. Antiphosphoserine immunoblotting revealed that sphingosine (10 microM for 3 min) increased the phosphoserine content of two proteins with approximate molecular masses of 40 and 70 kDa. Serine hyperphosphorylation of the 40-kDa protein was also observed in cells treated with EGF alone, whereas the intensity of the 70-kDa band was highest in cells treated with both sphingosine and EGF. The modulation of growth factor receptor-regulated signaling, including changes in [Ca2+]i, may constitute a mechanism by which elevations in cellular levels of specific sphingolipids, which occur transiently upon activation of certain receptors and chronically in sphingolipid storage diseases, exert their physiological and pathophysiological effects.