Myocardial ischemia alters the immunofluorescent staining pattern of lysosomal cathepsin D from one that is essentially participate to one in which a diffuse staining pattern predominates. Prolonged starvation causes an apparently similar redistribution of cathepsin D from discrete secondary lysosomes to diffusely staining sites. However, because of the limits of resolution of immunofluo-rescence techniques, it has not been possible previously to define the exact nature and location of cathepsin D staining in these conditions. Accordingly, a new procedure employing peroxidase-labeled antibodies has been developed to allow localization of cathepsin D with electron microscopy. Most of the cathepsin D that can be detected in normal hearts with this technique is present in classic lysosomal structures, analogous to the particulate staining pattern seen with immunofluorescence microscopy. The technique also reveals, for the first time, definitive ultrastructural evidence that ischemia induces lysosomal leakage and redistribution of cathepsin D into the cytoplasm. Starvation is not accompanied by release of cathepsin D into the cytoplasm; rather, there is widespread, increased distribution of cathepsin D within elements of the sarcoplasmic reticulum, whose diameters are too small for resolution with light microscopy. Thus, although both ischemia and starvation increase nonsedimentable cathepsin D activity, as measured biochemically, and produce a diffuse staining pattern under immunfluorescence microscopy, electron microscopy reveals a distinctly different subcellular distribution for this enzyme in the two conditions. These results emphasize that considerable caution must be exercised in interpreting biochemical and histochemical changes suggestive of enzyme release from lysosomes, in the absence of supportive ultrastructural evidence.Circ Res 46: 485-494, 1980