Abstract： AIM　To study the effects of dl-, l- and d-3-n-butylphthalide (NBP) on platelet aggregation and thrombus formation. METHODS　Thrombus formation was assessed by silk thread-induced thrombosis in arteriovenous shunt in rats. Rat platelet aggregation induced by adenosine diphosphate (ADP), arachidonic acid (AA), collagen and thrombin was detected in vitro. The generation of thromboxane B2 (TXB2) and the concentration of cAMP in rabbit platelets in vitro were studied with radioimmunological assay. RESULTS　dl-, l-NBP (5, 10, 20 mg*kg-1 ip) exhibited a dose-dependent inhibitory effect on thrombus formation in rats, while d-NBP was not active. dl, l, d-NBP (3-100 μmol*L-1) inhibited platelet-rich plasma aggregation in vitro induced by ADP, collagen and AA, and all of them showed no effect on thrombin-induced platelet aggregation. In addition, dl, l-NBP (10-100 μmol*L-1) were found to increase ［cAMP］i in dose-related fashion. In the meantime, only high concentration of l-NBP was found to decrease platelet TXA2 level. In addition, l-NBP (1-100 μmol*L-1) showed significant effect on inhibiting 5-HT release from platelets. In contrast, dl- and d-NBP showed no effect. CONCLUSION　The results suggest that NBP is a potent antiplatelet drug, the mechanism of its antithrombotic and antiplatelet activity is related to its regulation of cAMP level and 5-HT release.