The activity of four lysosomal enzymes (hyaluronidase, beta-N-acetylglucosaminidase, acid phosphatase, and cathepsin D) was studied in aqueous extracts of the light mitochondrial fraction of regenerating male rat liver. This tissue was chosen as a model for normal cell division in vivo. In the first wave of division, 40 to 50% of the cells divide synchronously. Activities were measured at 0, 9, 18 (end of G1 phase), 24 (S phase), and 30 hr (mitosis) and during regeneration, 4 and 11 days after partial hepatectomy. Activities were related to fresh tissue weight, to cellular DNA, and to protein content of the extracts. At 9 hr, there was an important increase in hyaluronidase and cathespin D activities (these two enzymes act upon macromolecules); beta-N-acetylglucosaminidase and acid phosphatase activities were only slightly increased. At the end of the G1 phase, 40 to 50% of the activity of all four enzymes was lost, which might indicate complete loss of activity in cells undergoing division. This depletion persisted until mitosis was complete. Four days later, there was a slow restoration of enzyme activities; after 11 days, hyaluronidase and cathepsin D exhibited about 80% of their initial activity, whereas beta-N-acetylglucosaminidase and acid phosphatase only regained about 50%. These results show that the lysosomal system perhaps plays some role in cell division.