The effects of cell-permeable C 2 and C 6-ceramides on human platelet responses were investigated. In thrombin-activated platelets, C 6 (5–30 μM) potentiated Ca 2+ mobilization and Ca 2+ influx, and decreased the rate of removal of Ca 2+ from cytosol. The effect of C 2 was not significant. Phorbol ester or calyculin A inhibition of thrombin-induced rises in platelet [Ca 2+] i was attenuated by C 6. Assays show that C 6 either prolonged the generation, or retarded the metabolism of inositol trisphosphates. Previous studies indicate that protein kinase C (PKC) acts in a negative feedback manner by inhibiting phosphatidylinositol breakdown, accelerating inositol trisphosphate metabolism, and increasing Ca 2+ pump activity. C 6 may counter these PKC effects indirectly. The synthetic ceramides inhibited platelet aggregation weakly and had no effect on pleckstrin (p47) phosphorylation. Recently we reported that C 2 but not C 6 inhibits superoxide generation and store-regulated Ca 2+ influx in neutrophils at similar concentrations. Cellular differences in ceramide metabolism or ceramide-sensitive enzymes and their substrates may account for the disparate results.