The cardiac lysosomal changes that accompany prolonged starvation and refeeding were studied using biochemical, immunohistochemical, and cytochemical techniques. Biochemical activities of lysosomal enzymes changed in a heterogeneous manner during starvation: cathepsin D increased significantly, acid phosphatase and β-glucosaminidase were altered minimally, and cathepsin B decreased significantly. During 1 to 3 days of refeeding, all activites returned toward normal except acid phosphatase. Morphological studies revealed that the increases in cathepsin D and acid phosphatase were localized in elements of the endoplasmic reticulum, Golgi complex, and secondary lysosomes; upon refeeding, these acid hydrolases were incorporated within autophagic vacuoles with simultaneous loss of staining within the endoplasmic reticulum. The results indicate that different cardiac lysosomal proteinases are not regulated in a coordinate manner during starvation. Moreover, changes in cathepsin D appear more likely to play an important metabolic role during the period of refeeding than during the period of starvation per se.