Studies were conducted to assess the effect of the serine protease inhibitor aprotinin on platelet adherence to both thrombin-stimulated and unstimulated human umbilical vein endothelial cells. Aprotinin treatment reduced significantly the adherence of platelets to endothelium pretreated or not with thrombin. In addition, aprotinin similarly reduced the adherence of platelets to plastic or collagen-coated tissue culture wells suggesting that the main site of action of the drug in this system is on the platelets. The role of endothelium-derived relaxing factor (EDRF; nitric oxide) in these platelet-endothelium reactions was investigated by prior incubation of both platelets and endothelial cells with NG-monomethyl-L-arginine (L-NMMA) which prevents the production of nitric oxide. The results demonstrated that nitric oxide was a significant inhibitor of the thrombin-induced platelet adherence in this assay system. Treatment with aprotinin in the presence or absence of L-NMMA reduced adherence of platelets to equivalent levels suggesting that aprotinin acts directly on the platelets via a mechanism that is EDRF-independent, to inhibit adherence.