An enzymatic assay for adenosine 3′:5′-monophosphate (cAMP) is described. Current measurement techniques can be expensive, time-consuming, and lack versatility. The critical step of this new method is the enzymatic destruction of endogenous purinergic noncyclic nucleotides. The diester linkage of cAMP is then cleaved and AMP is phosphorylated to ATP. Newly formed ATP is amplified using ATP-ADP cycling reactions and NADPH is measured fluorometrically. cAMP was measured in neonatal rat ventricular myocytes cultured on standard 100-mm dishes and treated with 2 μM 3-isobutyl-1-methylxanthine ± 1 μM isoproterenol. When the enzymatic fluorometric assay was compared with an immunocolorimetric assay and a radioimmunoassay, cAMP content (pmol/plate mean ± SE) was 124.3 ± 6.7, 130.6 ± 3.9, and 144.0 ± 4.4 without isoproterenol and 656.4 ± 23.5, 659.5 ± 54.1, and 677.1 ± 48.9 with isoproterenol, respectively. The standard curve with the enzymatic fluorometric assay is linear, in contrast to the curves of the nonlinear immunocolorimetric assay and radioimmunoassay. The enzymatic fluorometric assay can be used to detect <20 fmol of cAMP/sample and can be adapted to measure <1 fmol/sample. It can also be used to measure the activities of adenylate cyclase and phosphodiesterase. In summary, this enzymatic cAMP assay is sensitive, safe, versatile, and inexpensive and has multiple potential applications.