Carbachol (0.03–10 μM) or histamine (0.06–10 μM) challenge of indomethacin-pretreated rat aortic rings inhibited ADP-induced aggregation of platelets suspended in anticoagulated human whole blood. The maximum inhibition of platelet aggregation achieved with either drug was approximately 50%. No such inhibition was observed in rat aortic rings rubbed to remove endothelial cells or in intact vessels preincubated with nordihydroguaiaretic acid (NDGA, 10 μM), mepacrine (10 μM) or methylene blue (100 μM). Intravenously (i.v.) injected carbachol (0.5–5 μg/kg) also inhibited ADP-induced accumulation of 111indium-labelled platelets in the pulmonary circulation of urethaneanaesthetised rats. This effect of carbachol was inhibited by i.v. injected methylene blue (10 mg/kg) but unaffected by indomethacin (3 mg/kg) or hexamethonium (10 mg/kg) suggesting that PGI 2 or adrenaline release did not account for the inhibition of platelet activation observed. Similarly, the platelet inhibitory effect of carbachol in vivo was not related to increased pulmonary vascular blood flow (assessed by accumulation of 111indium-labelled erythrocytes) or blood fibrinolytic activity (measured by euglobulin clot bioassay). The present results suggest that endothelium-derived relaxing factor or a like substance inhibits human platelet aggregation in vitro and rat platelet aggregation in vivo.