The present study was undertaken to assess whether [32P]uptake and autoradiography could be used as an indicator of regional myocardial oxygen consumption. Rat hearts were perfused in the Langendorff mode with medium containing [32P]orthophosphate (32Pi) and the incorporation of label into ATP, perchloric acid-insoluble and soluble material and total tissue was determined. Oxygen uptake and perfusion pressure were continuously monitored. For control hearts the rate of incorporation of radioactivity into all four fractions was linear. Isoproterenol and pressure loading each increased total 32Pi uptake rate and the incorporation rate of 32Pi into ATP. Significant positive correlations were noted between the rates of total 32Pi uptake and oxygen uptake (r = 0.895; P < 0.001) and between the rates of 32P incorporation into ATP and oxygen uptake (r = 0.890; P < 0.001). Autoradiography of diffusible radioactive material (representing total 32Pi uptake) indicated that label correlated with oxygen uptake (r = 0.850; P < 0.001) and was distributed uniformly across the ventricle wall. Hearts subjected to 30 min of regional ischemia followed by reperfusion were indistinguishable from the control group in terms of either total 32Pi uptake or total oxygen uptake, but showed a marked necrotic area that was unlabeled surrounded by an area that was intensely labeled. It is concluded that autoradiography of diffusible radioactive material representing total 32Pi uptake may be applicable for assessing regional myocardial oxygen uptake. The technique has identified a region of tissue in reperfused ischemic hearts that surrounds necrotic tissue and which may have a compensatory increase in a oxidative metabolism.