The effect of acetylsalicylic acid on endothelial prostaglandin synthesis was measured in the presence of exogenous and endogenous substrates. In both types of measurement, a rate of inhibition was found similar to that observed for acetylsalicylic acid inhibition of cyclooxygenase activity in platelets. After withdrawal of acetylsalicylic acid, a rapid restoration of cyclooxygenase activity was observed when exogenous [1-14C]arachidonate was used as a substrate (50% of initial activity at 5 h). However, when endogenous substrate, released after phospholipase activation induced by thrombin treatment of the cells, was used to test cyclooxygenase activity, only partial restoration of enzymic activity was observed (30% after 48 h). Phospholipase activity, measured by the release of free fatty acids, was not inhibited by acetylsalicylic acid. Measurement of turnover times by incubating the cells with cycloheximide revealed a short turnover time for the enzymic activity tested with exogenous [1-14C]arachidonate (2.3 h) and a relatively long turnover time for the cyclooxygenase activity tested with endogenous substrate released after thrombin treatment of the cells (54 h). These results suggest that at least two pools of cycloxygenase are involved in endothelial prostaglandin synthesis.