Cardiac preparations from the right ventricular wall of rats were exposed to ATP-containing solutions of different Ca2+ concentrations ([Ca2+]) and Ca2+ buffering capacity to determine whether such treatments affected the Ca2+ handling properties of the cardiac muscle. Exposure to 8 mM extracellular ATP (ATP0) elicited sustained, near-maximal contractures that were due to Ca2+ entry from the extracellular environment and severely diminished cardiac excitability in a partially reversible manner. At 1 mM ATP0, there was a sustained rise in diastolic force and intracellular [Ca2+] and a marked, partially reversible change in the stimulation-induced Ca2+ transients measured with fura 2. A large, transient contracture was also observed when the intact ventricular preparations were transferred from the normal physiological solution to an ATP-containing (8 mM) relaxing solution (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid < or = 0.2 mM, < 10 nM Ca2+) with or without saponin (50 micrograms/ml). The ATP0-induced contractures were dependent on the prevalent conditions with respect to [Ca2+] and Ca2+ buffering capacity of the solutions and were associated with heavy Ca2+ loading of the sarcoplasmic reticulum (SR) and mitochondria, a less leaky SR, and a reduced SR sensitivity to the SR Ca2+ pump blocker 2,5-di-tert-butyl hydroquinone. The results indicate that the Ca2+ handling properties and the electrical excitability of ventricular preparations can be markedly modified by exposure to ATP0 and that conditions prevalent during skinning with saponin are important in determining the properties of the intracellular Ca2+ stores.