The modulation of the high-voltage-activated calcium current (ICa) by external ATP was examined in single ventricular cardiomyocytes of the hamster using the whole-cell configuration of the patch-clamp technique. Extracellular application of ATP (0.1-100 microM) was found to inhibit ICa reversibly. The inhibition followed a slow time course (half time approximately 25 s) and was accompanied by very small changes of the holding current and no shift in the current-voltage relationship. With 100 microM ATP, peak ICa was reduced by approximately 30%. This response was not blocked by the P1 inhibitor 8-cyclopentyl-1,3-dipropylxanthine. The nonhydrolyzable ATP analogs adenosine 5'-O-(3-thiotriphosphate) and AMP-adenosine 5'-[beta,gamma-imido]triphosphate also reduced ICa. The ATP analog alpha,beta-methylene-ATP was about equipotent with ATP at 50 microM. Internal guanosine 5'-O-(3-thiotriphosphate) (200 microM) rendered the ATP-mediated inhibition of ICa poorly reversible, whereas internal guanosine 5'-O-(2-thiodiphosphate) (200-500 microM) had no effect. Holding the intracellular adenosine 3',5'-cyclic monophosphate concentration at a constant high level did not alter the ATP response. We conclude that external ATP inhibits ICa via a P2 purinergic receptor in hamster ventricular myocytes. Our results suggest the involvement of a G protein not coupled to adenylate cyclase. The inhibition of ICa by extracellular ATP might have pathophysiological relevance under conditions of myocardial injury.