Treatment of Swiss 3T3 cells with sphingosine, a potential breakdown product of all sphingolipids, induced tyrosine phosphorylation of multiple substrates including bands of M(r) 110,000-130,000 and M(r) 70,000-80,000. Tyrosine phosphorylation in response to sphingosine occurred in a concentration dependent manner (EC50 = 10 microM) and developed gradually reaching half maximum and maximum effects at 20 and 60 min, respectively. The dihydroenantiomere of sphingosine, DL-threo-dihydrosphingosine, neither induced tyrosine phosphorylation nor interfered with sphingosine-stimulated tyrosine phosphorylation. Focal adhesion kinase (p125FAK) and paxillin were identified as prominent substrates for sphingosine-stimulated tyrosine phosphorylation. Cell permeable ceramides also stimulated tyrosine phosphorylation of the M(r) 110,000-130,000 band as well as p125FAK, but the effect was less pronounced than that of sphingosine. Tyrosine phosphorylation by sphingosine could be dissociated from both protein kinase C activation and Ca2+ mobilization from intracellular stores. Sphingosine stimulated striking actin stress fiber formation and focal adhesion assembly in Swiss 3T3 cells. The kinetics of actin stress fiber formation and tyrosine phosphorylation in response to sphingosine closely paralleled. Cytochalasin D, which disrupts the network of actin microfilaments, completely inhibited sphingosine induced tyrosine phosphorylation. In addition, tyrosine phosphorylation of p125FAK and paxillin in response to sphingosine was completely prevented when cells were stimulated in the presence of platelet-derived growth factor at a concentration (30 ng/ml) that caused disruption of the actin cytoskeleton. Our results demonstrate, for the first time, that sphingosine induces p125FAK and paxillin tyrosine phosphorylation, actin stress fiber formation and focal adhesion assembly in Swiss 3T3 cells.