A highly sensitive adenylate cyclase assay method has been developed using sequential chromatography on Dowex cation exchange resin and aluminum oxide. This method allows nearly complete separation of cyclic [32P]AMP from other [32P]-containing compounds, resulting in minimal radioactivity in assay blanks. The method is significantly more sensitive than previously reported methods, enabling detection of small amounts of cyclic AMP at low enzyme concentrations or early time points in kinetic studies. Adenylate cyclase, the enzyme that catalyzes the formation of cyclic AMP from ATP, can be assayed in vitro by measuring the production of radioactively labeled cyclic AMP from [α-32P]ATP. The challenge lies in separating cyclic AMP from the substrate and radioactive contaminants. Two methods are commonly used for isolating radioactive cyclic AMP: chromatography on Dowex resin followed by BaSO4 treatment, and one-step chromatography on aluminum oxide. Neither method completely eliminates [32P] in assay blanks. The authors report a highly sensitive adenylate cyclase assay combining Dowex resin and aluminum oxide chromatography, which nearly eliminates radioactivity in assay blanks. Materials include [α-32P]ATP, cyclic AMP, [3H]cyclic AMP, and various reagents. Methods involve preparing hepatic plasma membranes, determining protein content, and performing the adenylate cyclase assay with specific reagents and conditions. Isolation of cyclic AMP is done using three methods (A, B, and C), each with different chromatographic steps. Method C, which uses Dowex resin followed by aluminum oxide, provides the highest sensitivity with minimal blank values. Results show that Method C is the most sensitive, with the lowest blank values and highest cyclic AMP recovery. It allows measurement of basal activity at early time points, facilitating kinetic studies. Method C also requires less effort and time compared to other methods. The high sensitivity of Method C reduces the need for high radioactivity levels and allows lower concentrations of membrane protein in the assay. The study highlights the advantages of Method C for high sensitivity and efficiency in adenylate cyclase assays.A highly sensitive adenylate cyclase assay method has been developed using sequential chromatography on Dowex cation exchange resin and aluminum oxide. This method allows nearly complete separation of cyclic [32P]AMP from other [32P]-containing compounds, resulting in minimal radioactivity in assay blanks. The method is significantly more sensitive than previously reported methods, enabling detection of small amounts of cyclic AMP at low enzyme concentrations or early time points in kinetic studies. Adenylate cyclase, the enzyme that catalyzes the formation of cyclic AMP from ATP, can be assayed in vitro by measuring the production of radioactively labeled cyclic AMP from [α-32P]ATP. The challenge lies in separating cyclic AMP from the substrate and radioactive contaminants. Two methods are commonly used for isolating radioactive cyclic AMP: chromatography on Dowex resin followed by BaSO4 treatment, and one-step chromatography on aluminum oxide. Neither method completely eliminates [32P] in assay blanks. The authors report a highly sensitive adenylate cyclase assay combining Dowex resin and aluminum oxide chromatography, which nearly eliminates radioactivity in assay blanks. Materials include [α-32P]ATP, cyclic AMP, [3H]cyclic AMP, and various reagents. Methods involve preparing hepatic plasma membranes, determining protein content, and performing the adenylate cyclase assay with specific reagents and conditions. Isolation of cyclic AMP is done using three methods (A, B, and C), each with different chromatographic steps. Method C, which uses Dowex resin followed by aluminum oxide, provides the highest sensitivity with minimal blank values. Results show that Method C is the most sensitive, with the lowest blank values and highest cyclic AMP recovery. It allows measurement of basal activity at early time points, facilitating kinetic studies. Method C also requires less effort and time compared to other methods. The high sensitivity of Method C reduces the need for high radioactivity levels and allows lower concentrations of membrane protein in the assay. The study highlights the advantages of Method C for high sensitivity and efficiency in adenylate cyclase assays.