Isolated hearts subjected to ischemia-reperfusion (I/R) exhibit depressed cardiac performance and alterations in subcellular function. Since hearts perfused at constant flow (CF) and constant pressure (CP) show differences in their contractile response to I/R, this study was undertaken to examine mechanisms responsible for these I/R-induced alterations in CF-perfused and CP-perfused hearts. Rat hearts, perfused at CF (10 ml/min) or CP (80 mm Hg), were subjected to I/R (30 min global ischemia followed by 60 min reperfusion) and changes in cardiac function as well as sarcolemmal (SL) Na⁺-K⁺ ATPase activity, sarcoplasmic reticulum (SR) Ca²⁺-uptake and endothelial function were monitored. The I/R-induced depressions in cardiac function, SL Na⁺-K⁺ ATPase and SR Ca²⁺-uptake activities were greater in hearts perfused at CF than in hearts perfused at CP. In hearts perfused at CF, I/R-induced increase in calpain activity and decrease in nitric oxide synthase (eNOS) protein content in the heart as well as decrease in nitric oxide (NO) concentration of the perfusate were greater than in hearts perfused at CP. These changes in contractile activity and biochemical parameters due to I/R in hearts perfused at CF were attenuated by treatment with a NO donor, L-arginine, while those in hearts perfused at CP were augmented by treatment with L-NAME, an inhibitor of NO synthase. The results indicate that the I/R-induced differences, in contractile responses and alterations in subcellular organelles, between hearts perfused at CF and CP may partly be attributed to greater endothelial dysfunction in CF-perfused hearts than that in CP-perfused hearts.