Impaired platelet-derived nitric oxide (NO) contributes to acute coronary syndromes by enhancing platelet recruitment and thrombus formation. Polymorphic variants of the endothelial NO synthase (eNOS) gene have been associated with cardiovascular diseases. To examine whether eNOS variants affect platelet-derived NO and platelet function, and to assess the effects of estradiol on platelet function, we studied platelets from 47 healthy caucasians who were genotyped for eNOS polymorphisms in the promoter region (T-786 C), in intron 4, and in exon 7 (Glu298Asp). Platelet aggregation, platelet-derived NO and superoxide production were measured in control samples and samples pretreated with 17-alpha-estradiol (10 nmol/l). The occurrence of variants in the promoter region (P = 0.002) or in exon 7 (P = 0.007), but not in intron 4 (P > 0.05), were associated with lower levels of platelet-derived NO. An increased (P = 0.047) release of superoxide was observed with platelets from subjects with the variant in the promoter region, but not with other eNOS genetic variants. The eNOS gene polymorphisms did not affect ADP-induced platelet aggregation (P > 0.05). However, estradiol significantly increased platelet aggregation (P = 0.004), and platelet-derived superoxide (P = 0.047) in individuals homozygous for the variant in exon 7, but not in subject with other genotypes. These data demonstrate that the eNOS variants in the promoter region and in exon 7 decrease platelet-derived NO and that estradiol significantly increases platelet aggregation in homozygous for the variant in exon 7 but not in subjects with other genotypes, suggesting that eNOS variants may influence the thrombotic response.