Adenosine exerts a spectrum of energy preserving actions on the heart including negative chronotropic effects. The pathways leading to adenosine formation have remained controversial. In particular, although cytosolic 5'nucleotidases can catalyse adenosine formation in cardiomyocytes their contribution to the actions of adenosine has not been documented previously. We recently cloned two closely related AMP preferring cytosolic 5'nucleotidases (cN-IA and B); the A form predominates in heart. In this study we overexpressed pigeon cN-IA in neonatal rat cardiomyocytes using an adenovirus. cN-IA overexpression increased adenosine formation and release into the medium caused by simulated hypoxia and by isoproterenol in the absence and presence of inhibitors of adenosine metabolism. Adenosine release was not affected by an ecto-5'-nucleotidase inhibitor, ,-methyleneADP but was by the nucleoside transporter, dipyridamole. The positive chronotropic effect of isoproterenol (130±3 vs 100±4 beats/ min) was inhibited (107±3 vs 94±3 beats/ min) in cells overexpressing cN-IA and this was reversed by addition of adenosine receptor antagonist, 8-(p-sulfophenyl)theophilline (120±3 vs 90±4 beats/ min). Our results demonstrate that overexpressed cN-IA can be sufficiently active in cardiomyocytes to generate physiologically effective concentrations of adenosine at its receptors.