Compared to sham operated controls, myofilaments from hearts of ovariectomized ( OVX) rats demonstrate an increase in Ca²⁺ sensitivity with no change in maximum tension (Wattanapermpool and Reiser, Am J Physiol 1999;277:H467-73). To test the significance of this modification in intact cells, we compared intracellular Ca²⁺ transients and shortening of ventricular myocytes isolated from sham and 10 week OVX rats. There was a decrease in the peak Ca²⁺ transient with prolonged 50% decay time in OVX cardiac myocytes without changes in the resting intra-cellular Ca²⁺ concentration. Per cent cell shortening was also depressed and relaxation was prolonged in cardiac myocytes from OVX rats compared to shams. Ovariectomy induced a sensitization of the myofilaments to Ca²⁺. Hypercapnic acidosis suppressed the shortening of OVX myocytes to a lesser extent than that detected in shams. Moreover, a larger compensatory increase in % cell shortening was obtained in OVX myocytes during prolonged acidosis. The elevated compensation in cell shortening was related to a higher amount of increase in the amplitude of the Ca²⁺ transient in OVX myocytes. However, these differences in Ca²⁺ transients and % cell shortening were no longer evident in the presence of 1µM cariporide, a specific inhibitor of Na⁺/H⁺ exchanger type 1 (NHE1). Our results indicate that deprivation of female sex hormones modulates the intracellular Ca²⁺ concentration in cardiac myocytes possibly via an increased NHE1 activity, which may act in concert with Ca²⁺ hypersensitivity of myofilament activation as a determinant of sex differences in cardiac function.