The effects of 2-[(4-acetylphenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15), a synthetic 1,4-naphthoquinone derivative, on platelet activity and its mechanism of action were investigated. NQ-Y15 caused a concentration-dependent inhibition of the aggregation induced by thrombin, collagen, arachidonic acid (AA), and A23187. The IC₅₀ values of NQ-Y15 on thrombin (0.1 U/mL)-, collagen (10 μg/mL)-, AA (5C μM)-, and A23187 (2 μM)-induced aggregation were 36.2 ± 1.5, 6.7 ± 0.7, 35.4 ± 1.7, and 93.1 ± 1.4 μM, respectively. NQ-Y15 also inhibited thrombin-, collagen-, AA-, and A23187-stimulated serotonin secretion in a concentration-dependent manner. However, a high concentration (100 μM) of NQ-Y15 showed no significant inhibitory effect on ADP-induced primary aggregation, which is independent of thromboxane A₂ (TXA₂) production in rat platelets. In fura-2-loaded platelets, the elevation of intracellular free calcium concentration stimulated by AA, thrombin, and 4-bromo-A23187 was inhibited by NQ-Y15 in a concentration-dependent manner. The formation of TXA₂ caused by AA, thrombin, and collagen was inhibited significantly by NQ-Y15. NQ-Y15 inhibited TXA₂ synthase in intact rat platelets, since this agent reduced the conversion of prostaglandin (PG) H₂ to TXA₂. Similarly, NQ-Y15 selectively inhibited the TXA₂ synthase activity in human platelet microsomes, whereas it had no effect on activity of phospholipase A₂, cyclooxygenase, and PGI₂ synthase in vitro. NQ-Y15 inhibited platelet aggregation induced by the endoperoxide analogue U46619 in human platelets, indicating TXA₂ receptor antagonism, possibly of a competitive nature. These results suggest that the antiplatelet effect of NQ-Y15 is due to a combination of TXA₂ synthase inhibition with TXA₂ receptor blockade, and that it may be useful as an antithrombotic agent.