We have explored both the independent and combined effects of aspirin on cultured endothelial cells and platelets, and its influence on platelet deposition onto an extracellular matrix. Blood was circulated through a flat perfusion chamber with two coverslips placed sequentially with respect to blood flow. The first coverslip (upstream) was covered with a cultured endothelial cell monolayer, and the second (downstream) coated with extracellular matrix obtained after endothelial cell removal. Platelet interaction was measured on the second coverslip. Treatment of endothelial cells on the first coverslip with 100μM aspirin strongly reduced 6-keto-PGF1alevels recovered in the perfusates (118.3±35.8 vs 1038.0±308.5 pg/ml) and significantly increased platelet deposition on the downstream coverslip (% covered surface: 38.6±6.4% vs 14.6±1.8%;P<0.001). Increased platelet deposition (% covered surface: 24.9 f 3.1%;P<0.01) was observed in perfusions performed with blood containing aspirinized platelets, in the presence of intact endothelial cells. Treatment with aspirin of both platelets and endothelial cells had no additional effect on platelet adherence. Pretreatment of cultured endothelial cells with aspirin did not influence the adhesive properties of their underlying extracellular matrix. Our results indicate that, although endothelial cell cyclooxygenase is important in regulating platelet adhesion, its blockade seemed to have minimal effect on platelet deposition once platelet-cyclooxygenase was already inhibited.