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1. Adenylate cyclase activity in vascular endothelial cells (EC) has not been adequately defined. We compared adenylate cyclase activities in EC and smooth muscle cells (SMC) from the same specimen of fetal bovine aorta.2. The basal adenylate cyclase activities of EC and SMC did not differ significantly (18.9 ± 0.8 and 21.4 ± 1.7 pmol/mg protein per min, n = 10, respectively).3. The adenylate cyclase of EC responded dramatically to catecholamines, with the ED50 value for isoproterenol being 0.036 µmol/L, and was also more sensitive to calcitonin gene-related peptide than that of SMC.4. The adenylate cyclase of SMC was more sensitive to prostaglandins (with the ED50 for PGI2 being 0.024 µmol/L) and glucagon than that of EC, and responded modestly to catecholamines and was almost selectively sensitive to β1-adrenoceptor agonists. agonists.5. Maximum responses of adenylate cyclase to F-, guanosine 5′-o-(3-thiotriphosphate) and forskolin were greater in SMC than in EC.6. Based on these findings, it was concluded that EC and SMC differed significantly in adenylate cyclase responsiveness to agonists including hormones, prostaglandins, peptides and substances which may modify the effects of G proteins, although they shared a common developmental origin. Presumably, β-adrenoceptor agonists in EC and prostaglandins in SMC may appear to play an important role in cellular functions which are mediated by increases in cAMP.