During ischemia intracellular concentrations of inorganic phosphate (Pi) and hydrogen ions (H+) increase. Also, changes in myosin heavy chain isoform (MyHC) towards the -MyHC isoform have been reported following ischemia and infarction associated with coronary artery disease. The purpose of this study was to investigate the effects of myoplasmic changes of Pi and H+ on the loaded shortening velocity and power output of cardiac myocytes expressing either - or -MyHC. Skinned cardiac myocyte preparations were obtained from adult, male Sprague-Dawley rats (control or PTU treated to induce -MyHC) and mounted between a force transducer and servo-motor system. Myocytes were subjected to a series of isotonic force clamps to determine shortening velocity and power output during Ca²⁺ activations in each of the solutions: (i) pCa 4.5, pH 7.0, (ii) pCa 4.5, pH 7.0, 5 mM Pi, (iii) pCa 4.5, pH 6.6, and (iv) pCa 4.5, pH 6.6, 5 mM Pi. Added Pi and lowered pH each caused isometric force to decline to the same extent in -MyHC and -MyHC myocytes, however, -MyHC myocytes were more resistant to changes in absolute power output. For example, peak absolute power output fell 53% in -MyHC myocytes whereas power fell only 38% in -MyHC myocytes in response to elevated Pi and lowered pH (i.e., solution (iv)). The reduced effect on power output was the result of a greater increase in loaded shortening velocity induced by Pi in -MyHC myocytes and an increase in loaded shortening velocity at pH 6.6 that occurred only in -MyHC myocytes. We conclude that the functional response to elevated Pi and lowered pH during ischemia is MyHC isoform dependent with -MyHC myocytes being more resistant to declines in power output.