A sensitive and simple assay for adenosine 3′:5′-cyclic monophosphate (cAMP) has been developed based on competition for protein binding of the nucleotide to a cAMP-dependent protein kinase. The nucleotide-protein complex is adsorbed on a cellulose ester filter, and the assay is sensitive to 0.05–0.10 pmol of cAMP. This method is suitable for estimating cAMP levels in tissues, as it allows detection of as little as 0.05–0.10 pmol of cAMP in deproteinized tissue extracts. The binding constant approaches 10⁻⁹ M, and the assay is simple, accurate, and rapid. The method involves the use of a cAMP-dependent protein kinase from muscle, which has a favorable binding constant for cAMP. The binding of cAMP to specific proteins from Escherichia coli and adrenal cortex has been studied on cellulose ester filters. The binding of cAMP to specific proteins is quantitatively adsorbed on cellulose ester filters, and the assay can be used for unknown quantities of the cyclic nucleotide in deproteinized tissue extracts. The protein-kinase inhibitor preparation was modeled after that of Appleman et al. and was used to enhance the affinity of the cyclic nucleotide for the enzyme. The cyclic 3′:5′-nucleotide phosphodiesterase was purified from bovine cardiac muscle through the dialyzed acid-supernatant fraction of Butcher and Sutherland. The cAMP binding and cAMP assay involved the use of [³H]-cAMP and the protein-kinase inhibitor preparation. The binding reaction was conducted in a volume of 50 or 200 μl in 50 mM sodium acetate/acetic acid, pH 4.0, and incubated for longer than 60 min at 0°C. The binding protein was added, and the mixtures were diluted and filtered. The filter was washed and placed in a counting vial with Cellosolve. The scintillation mixture of toluene-Cellosolve (3:1) plus fluids was used, and the efficiency was approximately 30% (10,750 cpm/pmol). The results showed that the binding protein is the cAMP-dependent protein kinase, and the binding constant was evaluated from the data. The binding constant was 10–20 nM at pH 6 (phosphate), and a marked increase in affinity (Ka = 2–3 nM) was seen at pH 4. The inhibitor preparation increased the affinity approximately 2 times. The kinetics of the attainment of equilibrium at 0°C were shown, and the reverse reaction was studied by dilution and by the addition of a large excess of unlabeled cAMP. The reverse reaction was first orderA sensitive and simple assay for adenosine 3′:5′-cyclic monophosphate (cAMP) has been developed based on competition for protein binding of the nucleotide to a cAMP-dependent protein kinase. The nucleotide-protein complex is adsorbed on a cellulose ester filter, and the assay is sensitive to 0.05–0.10 pmol of cAMP. This method is suitable for estimating cAMP levels in tissues, as it allows detection of as little as 0.05–0.10 pmol of cAMP in deproteinized tissue extracts. The binding constant approaches 10⁻⁹ M, and the assay is simple, accurate, and rapid. The method involves the use of a cAMP-dependent protein kinase from muscle, which has a favorable binding constant for cAMP. The binding of cAMP to specific proteins from Escherichia coli and adrenal cortex has been studied on cellulose ester filters. The binding of cAMP to specific proteins is quantitatively adsorbed on cellulose ester filters, and the assay can be used for unknown quantities of the cyclic nucleotide in deproteinized tissue extracts. The protein-kinase inhibitor preparation was modeled after that of Appleman et al. and was used to enhance the affinity of the cyclic nucleotide for the enzyme. The cyclic 3′:5′-nucleotide phosphodiesterase was purified from bovine cardiac muscle through the dialyzed acid-supernatant fraction of Butcher and Sutherland. The cAMP binding and cAMP assay involved the use of [³H]-cAMP and the protein-kinase inhibitor preparation. The binding reaction was conducted in a volume of 50 or 200 μl in 50 mM sodium acetate/acetic acid, pH 4.0, and incubated for longer than 60 min at 0°C. The binding protein was added, and the mixtures were diluted and filtered. The filter was washed and placed in a counting vial with Cellosolve. The scintillation mixture of toluene-Cellosolve (3:1) plus fluids was used, and the efficiency was approximately 30% (10,750 cpm/pmol). The results showed that the binding protein is the cAMP-dependent protein kinase, and the binding constant was evaluated from the data. The binding constant was 10–20 nM at pH 6 (phosphate), and a marked increase in affinity (Ka = 2–3 nM) was seen at pH 4. The inhibitor preparation increased the affinity approximately 2 times. The kinetics of the attainment of equilibrium at 0°C were shown, and the reverse reaction was studied by dilution and by the addition of a large excess of unlabeled cAMP. The reverse reaction was first order