Sphingosine has been shown to be a potent and specific inhibitor of protein kinase C in vitro and in cell systems including human platelets. Questions have been raised as to the validity of commercial sphingosine as a protein kinase C inhibitor and whether sphingosine or N,N-dimethylsphingosine is the active species. In the present study, we compared the effects of synthetic D-erythro-sphingosine, N,N-dimethylsphingosine and commercial sphingosine on purified protein kinase C in vitro and washed human platelets. These three compounds were found to be of high purity and well-defined structure based on [1H]NMR, FAB-mass Spectrometry, and TLC analysis. Both synthetic D-erythro-sphingosine and commercial sphingosine inhibited protein kinase C in vitro using vesicle as well as mixed micellar assays. N,N-dimethylsphingosine also significantly inhibited purified protein kinase C in vitro. Both preparations of sphingosine inhibited phosphorylation for 40 kD protein, a known substrate of protein kinase C in platelets. Similarly both sphingosine preparations inhibited aggregation and secretion of human platelets induced by 8 nM gamma-thrombin. These results indicate that sphingosine from commercial source, synthetic sphingosine and N,N-dimethylsphingosine are equipotent in inhibiting protein kinase C. These studies also validate the utility of sphingosine as a phamarcologic inhibitor of protein kinase C in vitro and in cell systems.