The enzyme adenylyl cyclase has been shown to be important in the regulation of intraocular pressure. We therefore studied the activity of adenylyl cyclase (AC) activity in the rabbit iris/ciliary body (I/CB) after pre-treatment with theβ-adrenergic agonist isoproterenol (ISO) which activates cAMP dependent protein kinase A, and phorbol 12, 13 dibutyrate (PDB) which activates protein kinase C. When I/CB was pre-treated with ISO (1μM) or PDB (1μM), attenuated AC activity (∼35%) resulted when the activity of the enzyme was assessed by rechallenge with isoproterenol. However, when AC activity was assessed by rechallenge with forskolin or prostaglandin, enhanced activity resulted. In an effort to identify the mechanism of this apparent heterologous regulation of AC, studies were performed that showed no significant changes in the density ofβ-adrenergic receptors or the affinity of the receptors for the ligand (125I)-Iodopindolol occurred in ISO or PDB treated tissue. Similarly, in membranes prepared from ISO or PDB treated tissue, no significant changes in the functional activity of the guanine nucleotide binding proteins Gior Gscould be ascertained as assessed by somatostatin inhibition of forskolin-stimulated AC (to assess Gifunction), or in an adenylyl cyclase complementation assay (to assess Gsfunction). However, AC activity stimulated by Mn2+and purified Gswas enhanced (∼2X) following isoproterenol or phorbol ester pre-treatment, suggesting that an alteration at the level of the catalytic subunit of AC resulted from ISO or PDB pre-treatment. Therefore, the assessment of net changes in receptor coupled AC activity induced by phorbol esters or isoproterenol appears to be dependent on the drug used to rechallenge the AC system and cAMP production is dependent on the sum of diverse effects on multiple components of the AC pathway.