The authors have developed a highly sensitive adenylate cyclase assay method that combines sequential chromatography on Dowex cation exchange resin and aluminum oxide. This method, which uses [α-32P]ATP as the substrate, allows for the nearly complete separation of cyclic [32P]AMP from other 32P-containing compounds, significantly reducing background radioactivity. The method is more sensitive than previously reported methods, enabling the detection of small amounts of cyclic AMP at low enzyme concentrations or early time points in kinetic studies. The assay is particularly useful for evaluating the mechanism of hormone action through short-term kinetic studies. Method C, which involves passing the reaction mixture through a Dowex resin column before alumina chromatography, is found to be the most sensitive and efficient, with minimal background radioactivity and high cyclic AMP recovery. The method also allows for the reuse of columns, reducing time and effort. The high sensitivity of Method C can lower the required radioactivity and membrane protein concentrations, making it a valuable tool for further research.The authors have developed a highly sensitive adenylate cyclase assay method that combines sequential chromatography on Dowex cation exchange resin and aluminum oxide. This method, which uses [α-32P]ATP as the substrate, allows for the nearly complete separation of cyclic [32P]AMP from other 32P-containing compounds, significantly reducing background radioactivity. The method is more sensitive than previously reported methods, enabling the detection of small amounts of cyclic AMP at low enzyme concentrations or early time points in kinetic studies. The assay is particularly useful for evaluating the mechanism of hormone action through short-term kinetic studies. Method C, which involves passing the reaction mixture through a Dowex resin column before alumina chromatography, is found to be the most sensitive and efficient, with minimal background radioactivity and high cyclic AMP recovery. The method also allows for the reuse of columns, reducing time and effort. The high sensitivity of Method C can lower the required radioactivity and membrane protein concentrations, making it a valuable tool for further research.