The purpose of this study was to improve our previously described enzymatic fluorometric assay of the adenylate cyclase activity. Using physicochemical characteristics of NADPH, of which a 0.1 mmol/L solution would have an optical density of 0.627, we measured the adenylate cyclase activity by the spectral absorption of NADPH. The assay consists of two parts: pharmacological modulation of adenylate cyclase and measurement of newly synthesized cyclic AMP. The latter part involves four steps: enzymatic destruction of noncyclic adenine nucleotides and phosphorylated metabolites, conversion of cyclic AMP to ATP, amplification of ATP by enzymatic cycling, and measurement of NADPH with spectral absorption, which is generated in proportion to initial cyclic AMP levels. This new assay was tested in membrane preparations made from rat hearts in comparison with the previously described fluorometric assay. We obtained identical results by spectrophotometry and fluorometry with high reproducibility. Because the fluorometric assay possesses a high sensitivity while the spectrophotometric method is advantageous because of its wide analytical range of cyclic AMP measurement, combination of fluorometric and spectrophotometric methods may offer a convenient way to measure the adenylate cyclase activities in various samples.