These studies were undertaken to examine the effects and the mechanism of action of flurazepam and diazepam on human platelet activation. One minute preincubation with flurazepam (3–300 uM) or diazepam (3–300 uM) inhibited platelet aggregation, serotonin secretion and prostaglandin synthesis induced by ADP (1–5 uM), epinephrine (1–5 uM), and arachidonic acid (600–1000 uM). However, 357% higher concentration of diazepam (265 uM) as compared to flurazepam (58 uM), was required to inhibit arachidonic acid induced production of malondialdehyde (MDA) by 50%. In addition, flurazepam and not diazepam inhibited the release of arachidonic acid from platelet phospholipids in a concentration dependent manner. In other experiments flurazepam but not diazepam also blocked aggregation and secretion induced by U46619 (2 uM), a stable analog of prostaglandin H2. Platelet aggregation and serotonin secretion induced by collagen (40–300 ug/ml) was inhibited by flurazepam with an IC-50 of 153 uM and 136 uM respectively, whereas higher than 300 uM diazepam was required to inhibit collagen-induced aggregation and secretion by 50%. Flurazepam and diazepam both exhibited their most potent antiplatelet effects against phospholipase C-induced aggregation which is mediated by prostaglandin-independent mechanisms. Only 15 uM and 11 uM flurazepam and 31 uM and 27 uM diazepam were needed to inhibit PLC-induced aggregation and secretion of serotonin by 50% respectively. Effects of these benzodiazepines on platelet cyclic AMP and cyclic GMP were also examined. Neither flurazepam nor diazepam caused any significant change in cyclic AMP or cyclic GMP levels in platelets.. These findings suggest that: (a) flurazepam, as compared to diazepam, is 106% - 357% more effective in inhibiting platelet aggregation and serotonin secretion induced by arachidonic acid , collagen and phospholipase C; (b) flurazepam inhibits platelet activation by inhibiting the release of arachidonic acid, its conversion into prostaglandins and by blocking the action of prostaglandins on platelets; (c) diazepam does not inhibit thrombin-induced release of arachidonic acid, conversion of exogenously added arachidonic acid into MDA, or the action of prostaglandins; (d) both flurazepam and diazepam inhibit PLC-mediated activation of platelets; and (e) neither diazepam nor flurazepam achieve their antiplatelet actions by affecting platelet cyclic nucleotide levels.