OBJECTIVE To characterize in vivo changes of adenylate cyclase activity in rabbit ciliary processes during the circadian cycle. METHODS After removal of vitreous, lens, retina and choroid from freshly enucleated rabbit eyes, the anterior segment and attached sclera was chilled in cold buffer containing the non-selective phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Then ciliary processes were excised and homogenized in ice cold trichloro-acetic acid (TCA) 2.5 min after IBMX treatment. Increased cyclic AMP in response to PDE inhibition was measured in ciliary processes at six times during the circadian cycle, after preganglionic section of the cervical sympathetic trunk (decentralization, DX), and after topical instillation of hydroxyamphetamine, timolol, brimonidine, rauwolscine or a soluble derivative of forskolin. RESULTS The increase of rabbit ciliary process cyclic AMP levels in response to PDE inhibition with IBMX was enhanced at night. Much of the enhanced nocturnal increase persisted in constant dark and was blunted by DX. Topical instillation of hydroxyamphetamine enhanced the increase during the day; whereas, timolol, a beta-adrenergic antagonist, or brimonidine, an alpha2-adrenergic agonist, blunted the IBMX induced increase. Neither instillation of rauwolscine, an alpha2-adrenergic antagonist, nor the soluble forskolin derivative enhanced the increase of cyclic AMP after IBMX. CONCLUSIONS The technique reported here can be used to estimate ciliary process adenylate cyclase activity in vivo. There is a circadian rhythm of adenylate cyclase activity in rabbit ciliary processes that is driven in part by ocular sympathetic input and stimulation of beta-adrenergic receptors.