Oxygen diffusion in plasma membranes and mitochondrial (Mt) membranes of ventricular myocytes isolated from adult rat hearts was measured using O2-induced fluorescence quenching of pyrene-butyric acid. The diffusion coefficient for oxygen (DO2 in 10(-6).cm2 x s-1) of the plasma membrane was 2.91 +/- 0.05 (37 degrees C) in the control group, which was significantly higher than that of Mt membrane (2.23 +/- 0.11) (P < 0.001). The DO2 of the plasma membrane was reduced to 2.50 +/- 0.08 (P < 0.001) in myocytes isolated from heart after 15 min reperfusion following no-flow ischemia for 30 min, while that of the Mt membrane remained almost unchanged. Administration of hydrogen peroxide and hypochlorous acid to the isolated myocytes also reduced the plasma membrane DO2. The decrease in the plasma membrane DO2 correlated with that in the fraction of rod-shaped myocytes. We conclude that the plasma membrane is more susceptible to reperfusion injury than the Mt membrane, but these membranes do not limit O2 uptake in the heart because of the high absolute values of DO2.