Abstract A double-isotope dilution and derivative analysis technique is described for the measurement of adenosine 3′,5′-monophosphate. Column and paper chromatography are used for the isolation of adenosine 3′,5′-monophosphate from tissues and urine. The nucleotide is esterified with tritium-labeled acetic anhydride via an acetylimidazole intermediate to form adenosine 3′,5′-monophosphate 2′-acetate. Specificity of the technique was demonstrated by repeated chromatography of samples and standards with maintenance of constant specific activity and also by comparison with other techniques using single-isotope dilution analysis. Recovery studies and analysis of proportional quantities of tissues are also presented as evidence of specificity. Technique precision was demonstrated by a coefficient of variation of 7.5% in measurements of 3 × 10 −9 mole of liver adenosine 3′,5′-monophosphate. The practical sensitivity of the technique is 5 × 10 −10 mole. Values for several rat tissues are presented.