Injection of leupeptin (an inhibitor of lysosomal cathepsins B, H, and L) to nonstarved rats causes an expansion of the autophagic vacuolar (AV) system in hepatocytes. Readily identifiable cytoplasmic constituents were seen within the AVs shortly after the administration. Later, the contents of the AVs seemed to reach more advanced stages of degradation. Liver AVs were purified by a one-step centrifugation of a crude mitochondrial lysosomal fraction in a discontinuous metrizamide gradient after exposing the rats to leupeptin for varying periods of time. Leupeptin caused alterations of the AV fraction that were time dependent. Initially, i.e., after 30 minutes of leupeptin exposure, mature (secondary) lysosomes clearly dominated over nascent AVs. The situation was reversed when fractionation was performed 1 or 2 hours following the injection of leupeptin. Now, the AVs were more frequent than the mature lysosomes. Later, the proportion of mature lysosomes was again larger. An increase in dense bodies was noted after 16 hours of leupeptin treatment. The proteolytic capacity of the AVs at different stages of maturation was measured after labeling liver proteins with an injection of L-1-14C-leucine 16 hours before sacrifice. AVs were purified after varying times of exposure to leupeptin. The proteolysis decreased greatly 1 to 2 hours following the injection of leupeptin but never ceased. On the other hand, lipolysis seemed unaffected by leupeptin using a similar experimental protocol as for proteolysis. If the animals were subjected to more lasting exposure to leupeptin before fractionation, proteolysis increased, displaying a peak higher than control, occurring after approximately 4 hours. The degradation gradually returned to control values after 16 hours. A catch-up in proteolysis was thus observed. The time course of proteolysis was reflected in the protein content in the AV fraction. After an initial increase that coincided with the lowered proteolysis, it returned to control level. Marker enzyme activities for endoplasmic reticulum and mitochondria (G6Pase and succinate-cytochrome c reductase) followed the same pattern. The AV content of the cytosolic enzymes lactate dehydrogenase and aldolase reached as high as 2.30 and 2.80% of the values in the homogenate during the 1st hour of leupeptin exposure. From these data the half-lives of the enzymes were calculated. They were: for aldolase, 43 hours; for LDH, 68 hours. This suggests that AVs account for a substantial proportion of degradation not only of organelles but also of soluble cytosolic enzymes.