Isolated rat heart cells permeabilized by digitonin were examined as an experimental model to study heart bioenergetics. The cells displayed good parameters of oxidative phosphorylation (acceptor control ratio ca. 8 with pyruvate + malate). High ATPase activity detected in the cells was characterized. The ATPase activity was Ca-dependent (optimum [free Ca] ca. 400 nM), and Mg was necessary for its full activity. Double reciprocal plot l/v vs. 1/[free Ca] at physiological [free Ca] was linear, thus showing free Ca to be a substrate for the ATPase (Km for Ca ca. 149 nM). Double reciprocal plot 1/v vs. 1/[ATP] was also liner, thus showing the ATPase activity could be ascribed to a single enzyme. The ATPase is supposed to represent Ca, Mg-ATPase of sarcoplasmic reticulum. The ATPase activity appeared to be functionally coupled to oxidative phosphorylation of the cells by apparently preferring ATP supplied by mitochondria (KmATP = 74 microM) to external ATP (KmATP = 169 microM; P less than 0.05). Apparent preference by oxidative phosphorylation for ADP supplied by the ATPase (KmATP = 45 microM) to external ADP (KmATP = 152 microM) was also manifested by significant difference (P less than 0.02) in Km.