Previous studies from our laboratory have shown that sphingosine (Zhang et al. (1990) J. Biol. Chem. 265, 76-81) and sphingosine 1-phosphate (Zhang et al. (1991) J. Cell. Biol. 114, 155-167), metabolites of membrane sphingolipids, stimulate release of calcium from internal sources and increase proliferation of quiescent Swiss 3T3 fibroblasts acting in a fundamentally different, protein kinase C-independent pathway. The mitogenic effect of sphingosine was accompanied by an increase in the levels of phosphatidic acid (PA), a potent mitogen for a variety of cell types, that may function as an intracellular second messenger (Zhang et al. (1990) J. Biol. Chem. 265, 21309-21316). Sphingosine also induced early increases in sphingosine 1-phosphate (SPP) levels that preceded the increase in PA (Desai et al. (1992) J. Biol. Chem. 267, 23122-23128). SPP itself produced a more rapid increase in PA, thus suggesting that it may mediate the effects of sphingosine on PA accumulation. The concentration dependence for the formation of PA induced by SPP correlated with its effect on DNA synthesis. Similar to sphingosine, SPP also stimulated the activity of phospholipase D, although a significant effect was observed at a much lower concentration. However, in contrast to previous reports with sphingosine, SPP did not inhibit the PA phosphohydrolase activity in cell homogenates. Thus, in addition to its effect on mobilization of calcium, SPP can increase the level of PA, most likely via activation of phospholipase D. We suggest that SPP mediates the effect of sphingosine on PA accumulation in Swiss 3T3 fibroblasts and may regulate cellular proliferation by affecting multiple transmembrane signaling pathways.