A CE assay was developed for the detection of adenylyl cyclase (AC) activity stimulated at the AC and G protein-coupled receptor (GPCR) level. In the assay, cell membranes overexpressing GPCR and/or AC were incubated with modulators and substrate ATP to produce cAMP in a dose-dependent manner. In both the CE-UV and a radiochemical assay, the addition of forskolin (FSK) resulted in a two- to three-fold maximum increase in AC activity with EC(50)s of 4.2 +/- 0.7 and 2.4 +/- 0.7 mu M, respectively, demonstrating that similar results were obtained by both assays. GPCR activation was also detected using cell membranes overexpressing AC and the beta(2)-adrenergic receptor AAR) fused to the stimulatory G protein. Terbutaline (beta(2)AR agonist) increased the basal rate of cAMP formation 1.7 +/- 0.1-fold resulting in an EC50 of 62 +/- 10 nM. The assay's ability to detect antagonists is demonstrated by the expected right-shifted EC50 of terbutaline by the beta(2)AR antagonist propranolol. The CE-UV assay offers advantages over the traditional radioactivity assay in terms of safety and labor.