Platelet aggregation plays an important role in haemostasis and vascular disorders. This mainly takes place by the action of endogenous agonists like ADP, platelet-activating factor (PAF), epinephrine, 5-hydroxytryptamine (5-HT) and arachidonic acid (AA). These agonists except AA interact with G-protein coupled receptors Recent studies have shown that phosphatidylinositol 3-kinase (PI 3-kinase) and myosin light chain kinase (MLCK) play an important role in platelet aggregation We have shown that low doses of the selective PI 3-kinase inhibitor, wortmannin, inhibits aggregation (IC50; 20 nM) mediated by subthreshold doses of 5-hydroxytryptamine (5-HT; 5 mu M) and epinephrine (1 mu M). This study was undertaken to examine the effects of wortmannin in platelet aggregation induced by various platelet agonists. The results show that wortmannin inhibited aggregation mediated by various agonists with an IC50 for ADP (110 nM), AA (2 mu M), collagen (280 nM) and PAF (520 nhl). These studies suggest that wortmannin-mediated inhibition of aggregation induced by ADP, epinephrine, collagen and PAF may affect multiple enzymatic pathways and the most likely targets seem to be both PI 3-kinase and MLCK as other kinases like cAMP- and calcium-calmodulin-dependent protein kinases are reported to be not affected by wortmannin.