The infiltration of neutrophils into ischemic myocardium exacerbates myocardial damage upon reperfusion, whereas drugs that inhibit neutrophil activity or function reduce infarct size. Consequently, it is important to accurately assess the myocardial neutrophil content. Histologic sections and radiolabeled cells have been used, but have major limitations. We have developed a method to measure the neutrophils present in cardiac tissue by utilizing a spectrophotometric assay for the neutrophil-specific myeloperoxidase enzyme (MPO) (Bradley et al., 1982a). Coronary artery occlusion and reperfusion in the anesthetized dog induces neutrophil accumulation into the ischemic heart, which shows a linear relationship with time. An increase in activity from 0.014 +/- 0.001 units (u) MPO/100 mg tissue to 0.091 +/- 0.02 u MPO/100 mg is already apparent at the end of the 90-min occlusion period. This activity increases over 5 hr reperfusion to 0.32 +/- 0.018 u MPO/100 mg tissue. Histologic analyses confirmed the temporal association of neutrophil accumulation. Moreover, there is a correlation between infarct size and tissue MPO activity. Measuring the MPO content in preparations of canine neutrophils, which is directly correlated with cell number, allows units of MPO activity to be converted into a tissue neutrophil content. This assay is simple, sensitive, and provides a quantitative index of myocardial neutrophil accumulation that can be used to study the relationship between leukocyte infiltration and myocardial injury.