Adenosine and adenine nucleotides shorten the action potential duration of atrial myocytes and activate a specific acetylcholine and adenosine receptor-operated potassium outward current referred to as IKACh,Ado*The objective of this study was to determine whether adenine nucleotides shorten the action potential duration and increase IKach,Adoin guinea pig atrial myocytes by directly activating adenosine receptors. The potency and efficacy of AMP and adenosine in increasing IKACh,Adoand shortening atrial action potential duration were similar; the EC50values for AMP and adenosine were 3.4±0.8 and 3.1±0.4 μM, respectively. Likewise, the maximum increases in Ikach,Adocaused by AMP and adenosine were similar (122±11% versus 123±9%). In comparison, ATP and the stable analogue of AMP, adenosine monophosphorothioate (AMPS), were significantly less potent and efficacious than adenosine and AMP, and inosine had no effect. The electrophysiological effects of AMP were antagonized by the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline and abolished in the presence of adenosine deami-nase and α,β-methylene-ADP (APCP, an inhibitor of AMP degradation). Binding of the A1-adenosine antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX) to guinea pig atrial membranes treated with adenosine deaminase and APCP was reduced up to 60% by 100 μM concentrations of AMP, AMPS, and adenosine. Inosine inhibited binding by 43±3% at 100 μM, whereas hypoxanthine and xanthine had little (5–10% inhibition) and uric acid had no effect. Only 3% of AMP and 35% of AMPS were recovered intact after a 90-minute incubation at 21°C with preparations of guinea pig atrial membranes. Percent displacement of [3H] DPCPX binding to atrial membranes by 100 μM AMP was significantly less in the presence of nucleoside phosphorylase and xanthine oxidase (to degrade inosine, hypoxanthine, and xanthine to uric acid) than in their absence (12.4±3.1% versus 49.7±1.5%). The results suggest that the observed electrophysiological actions of adenine nucleotides in cardiomyocytes are mediated by adenosine and are consistent with activation of A1-adenosine receptors.