In heart membranes, specific [3H](+)-isradipine binding is reduced in membranes from ischemic hearts and by adding 1 mM ATP at low Ca2+ concentrations (1 microM). We investigated if ATP affected specific [3H](+)-isradipine binding in intact rat ventricular cardiomyocytes. Reducing intracellular ATP by 2 h hypoxia (N2 gas) and glucose-free buffer with 1 mM CN-, did not affect density or dissociation constant of [3H](+)-isradipine binding in cardiomyocytes at extracellular 30 mM K+. Extracellular 10 mM ATP inhibited binding in cardiomyocytes by 90% and 50%, respectively, in 30 mM and 120 mM K+ buffer with Ca2+ and Mg2+. Omitting Ca2+ and Mg2+ from the buffer had no effect on the binding inhibition of ATP. Hence, in cardiomyocytes, reducing intracellular ATP has no effect on specific [3H](+)-isradipine binding, whereas high extracellular ATP in the presence of Ca2+ and Mg2+ inhibits binding. Apparently, ATP effects on binding differ in cardiomyocytes and membranes.