Aggregation and serotonin secretion were studied in washed rat platelets after oral administration of ticlopidine or its more potent analog PCR 4099. Besides a complete suppression of the ADP-induced aggregation, the two drugs significantly inhibited aggregation and secretion induced by three protein kinase C activators (1-oleoyl-2-acetyl-sn-glycerol, OAG; 12-0-tetradecanoyl phorbol-13-acetate, TPA; phospholipase C), by the calcium ionophore A 23187 and by thrombin. The highest inhibition was observed at low stimuli concentrations but could be partly or almost completely overcome by increasing their concentrations. The combination of aspirin (ASA) with the ADP scavenging system, creatine phosphate/creatine phosphokinase (CP/CPK) in vitro resulted in an inhibition similar to that observed ex vivo after ticlopidine or PCR 4099 treatment. Moreover, these in vitro and ex vivo treatments were not additive. As identical results were obtained with CP/CPK alone but not with ASA, it is concluded that ticlopidine and PCR 4099 do not interfere with protein kinase C or calcium movements but specifically inhibit the effects of released ADP, which might explain the broad spectrum anti-platelet activity of these drugs.