Na+/Pi-cotransporter utilizes the electrochemical gradient of sodium to transport inorganic phosphate (Pi) into the cells of various organs, including heart. Because changes in the activity of Na+/Pi-cotransporter may influence the concentrations of intracellular Na+, Ca2+ and other ions, the influence of Pi on myocardial contractility was investigated. Interaction between Pi and ouabain was also investigated. Experiments were performed using isolated perfused rat heart under conditions of constant preload (15 mm Hg) and controlled calcium activity (0.717 mM). The electronically differentiated value of the left ventricular pressure (LVP) signal was used as an index of myocardial contractility. When the heart was treated with 2.5 mM Pi, myocardial contractility increased slightly. Contractility was increased significantly by treatment with 5 mM Pi. In the presence of 10 mM Pi, the heart showed marked but transient increase in contractility for the first 15 min of the 60-min treatment period. Phosphonoformate (PFA), an inhibitor of Na/Pi-cotransporter, showed selective inhibition of Pi-induced increase in myocardial contractility. In another study, ouabain and Pi showed additive effect on contractility. The data from these studies suggest that myocardial contractile responses to Pi depend on Pi concentration and duration of Pi treatment. The data also support the conclusion that Na+/Pi-cotransporter may play a role in myocardial contractility.