Lipopeptide analogues of the NH2-terminus of bacterial lipoprotein are known to induce activation of macrophages, neutrophils, and lymphocytes. We studied the effect of the lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-( S)-seryl-(S)-lysyl-(S)-lysyl-(S)-lysyl-(S)-lysine [Pam3CysSer(Lys)4] on several functions of human platelets. Pam3CysSer(Lys)4 led to the aggregation of platelets and induced the secretion of serotonin with an effectiveness similar to thrombin. These cellular effects of Pam3CysSer(Lys)4 were concentration dependent, being half maximal at 2-3 microM and maximal at 10-30 microM. Another lipopeptide also induced platelet aggregation and serotonin secretion but was less potent and less effective than Pam3CysSer(Lys)4. The lipid moiety and the peptide moiety of Pam3CysSer(Lys)4 alone were without any effect. Lipopeptides also stimulated tyrosine phosphorylation of several proteins with molecular masses similar to those found to be tyrosine phosphorylated in response to thrombin, and Pam3CysSer(Lys)4 led to an increase in the cytosolic calcium concentration. All studied responses of platelets to lipopeptides were inhibited by the prostacyclin receptor agonist cicaprost. Taken together, our data show that lipopeptides are effective activators of human platelets and that this activation is susceptible to the action of physiological platelet inhibitors.