TPA regulation of prostaglandin H synthase activity in primary and subcultured dog urothelial cells was investigated. Previous studies have demonstrated an early (0-2 hr) increase in PGE, synthesis mediated by TPA which is dependent upon release of endogenous arachidonic acid by a phospholipase-mediated pathway. in this study, prostaglandin H synthase activity was assessed directly with microsomes and indirectly after addition of exogenous arachidonic acid at a maximum effective concentration (100 pM) to media. PGE, synthesis, measured by radioimmunoassay, served as an index of prostaglandin H synthase activity. After a 24-hr incubation with 0.1 pM TPA or 1 .O pM A23187, arachidonic acid elicited significantly more PGE, synthesis in agonist-treated cells than it did in control cells in primary culture. Microsomes from 24-hr TPA-treated cells exhibited significantly more prostaglandin H synthase activity than did those from control cells. In addition, the PGE, content of overnight media was approximately lo-fold greater in TPA-treated cells than in control cells. The late (24 hr) response was more sensitive to lower concentrations of TPA than was the earlier (0-2 hr) response. TPA at 0.1 pM was a maximum effective dose for both responses. The 24-hr response was blocked by cycloheximide and staurosporine, inhibitors of protein synthesis and protein kinase C, respectively. Pretreatment of cells with aspirin, an irreversible inhibitor of prostaglandin H synthase, prior to addition of TPA did not prevent the late TPA-mediated increase in PGE, synthesis. Subcultured cells exhibited both an early and a late TPA response. Only the early response was inhibited by aspirin pretreatment. Results suggest that the late response with TPA is caused by de novo synthesis of prostaglandin H synthase. Thus, primary and subcultured dog urothelial cells possess two distinct mechanisms for regulating signal transduction by arachidonic acid metabolism. This study provides a basis for assessing these mechanisms of signal transduction in urothelial cell lines and transformed cells