Evidence suggests ischemia/reperfusion injury is largely due to cytosolic Ca accumulation resulting from functional coupling of Na/Ca exchange (NCX) with stimulated Na/H exchange (NHE1). Evidence also suggests 17-estradiol (E2) stimulates release of NO which inhibits NHE1. Thus we tested the hypothesis that acute E2 limits myocardial Na and therefore Ca accumulation and thereby limits ischemia/reperfusion injury. NMR measured cytosolic pH (pH_i), Na (Na_i) and calcium concentration ([Ca]_i) in Krebs-Henseliet-perfused hearts from ovariectomized rats (OVX). Left ventricular developed pressure (LVDP) and lactate dehydrogenase (LDH) release were also measured. Control ischemia/reperfusion was 20 min of baseline perfusion, 40 min global ischemia, and 40 min of reperfusion. The E2 protocol was identical except 1 nM 17-estradiol was included in the perfusate prior to ischemia and during reperfusion. Data are expressed as mean±SEM and the following differences were found, p<0.05. E2 limited the changes in pH_i, Na_i, and [Ca]_i during ischemia. For control OVX vs. OVX+E2: pH_i fell from 6.93±0.03 to 5.98±0.04 vs. 6.96±0.04 to 6.68±0.07; Na_i (mEq/kg dry wt) rose from 25±6 to 109±14 vs. 25±1 to 76±3; [Ca]_i (nM) changed from 365±69 to 1248±180 vs. 293±66 to 202±64. E2 also improved recovery of LVDP and diminished release of LDH during reperfusion. Effects of E2 were diminished by 1 µM N-nitro-L-arginine methyl ester (LNAME). Thus, the data are consistent with the hypothesis. However, E2 limitation of increases in [Ca]_i is greater than can be accounted for by the thermodynamic effect of reduced Na_i accumulation on NCX.