We have used a new technique for extraction of myocardial membranes (0.25 M sucrose, 0.6 M KC1) to isolate particulate and soluble proteins and enzymatic activities in an effort to quantify changes characteristic of progressive ischemia. Myocardial blood flow (MBF) was measured with microspheres (15 fiia diameter) in all samples of tissue used for assay of proteins and enzymatic activities; MBF to the moderately ischemic areas (M-ischemia) was 53% of control (H-control); MBF to the severely ischemic areas (L-ischemia) was 9% of control. Significant decreases (P< 0.001) in content of protein were seen in all post 1,000 g pellets and supernatant fluids in the L-ischemia zones; particulate lysosomal enzymatic activity was significantly decreased (P < 0.001) in all four post 1,000 g pellets (2,500 g to 140,000 g) of the L-ischemic areas (for N-acetylβ-glucosaminidase and β-glucuronidase). The increase in percent free activity of lysosomal enzymes (index of loss of latency) also was highly significant (P < 0.001) in all particulate fractions of the L-ischemic areas. In addition, about 45% of the total activity of the microsomal marker enzyme, rotenone-insensitive NADH cytochrome C reductase (RINCR), was found in the 140,000 g pellet of H-control tissue (9.9 μmol/min per g); this activity fell to 8.1 μmol/min per g in M-ischemic areas (P < 0.001) and to 5.3 μmol/min per g in L-ischemic areas (P < 0.001). This study demonstrates that changes in myocardial proteins, lysosomes, and other membrane-bound enzymes (RINCR) may provide reproducible bichemical parameters for assessing ischemic myocardial injury.