We have earlier reported that neutrophils play an important role in pulmonary thromboembolism. The effect of circulating polymorphonuclear leukocytes (PMNL) isolated from normal and thrombotic rats was, therefore, studied on platelet aggregation in platelet-rich plasma. PMNL inhibited the platelet aggregation in a concentration- and time-dependent manner at 37-degrees-C. The platelet aggregation inhibitory response of PMNL was more pronounced in the presence of superoxide dismutase and was abolished by hemoglobin and methylene blue. These observations suggested that the inhibitory effect of PMNL on platelets was mediated by neutrophil-derived relaxing factor. PMNL, obtained after thromboembolism, inhibited the platelet aggregation response more strongly due to an increased release of nitric oxide from them. It is suggested that PMNL play an important role in the regulation of hemostasis.