Adenylate cyclase, the catalytic protein that converts ATP to cAMP, plays a fundamental role in adrenergic signal transduction. Adenylate cyclase activity (pmol cAMP/mg/min) is generally assayed by measuring radiolabeled cAMP generated from [α-32P]ATP. Although sensitive, the radioactive approach is costly and time consuming. Given safety and environmental concerns, we developed a highly sensitive fluorometric assay for adenylate cyclase activity. This assay depends upon the breakdown of cAMP by phosphodiesterase to AMP, and the subsequent stimulation by AMP of glycogen phosphorylase a. Radioactive and fluorescence methods were compared using the same ventricular membrane preparations from five different rabbit hearts. Theophylline, used in the fluorometric assay, increased basal adenylate cyclase activity. However, adenylate cyclase kinetics, the dose response to isoproterenol, and the "fold" stimulation (agonist stimulated/basal adenylate cyclase activity) after isoproterenol (10-6M), guanylyl-5′-imidodiphosphate (GppNHp) (10-4M), and NaF (10-2M) were nearly identical with both methods. Adenylate cyclase activity can be measured with the fluorometric assay in samples as small as 10 μg of membrane protein. In summary, this new fluorometric assay is highly sensitive, safer, less costly, and less time consuming than radioactive assays for adenylate cyclase activity.