The activity of 4 lysosomal enzymes (hyaluronidase [EC 3.2.1.35], .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, 40-50% of the cells divide synchronously. Activities were measured at 0, 9, 18 (end of G1 phase), 24 (S phase), and 30 h (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 h, there was an important increase in hyaluronidase and cathepsin D activities (these 2 enzymes act upon macromolecules); .beta.-N-acetylglucosaminidase and acid phosphatase activities were only slightly increased. At the end of the G1 phase, 40-50% of the activity of all 4 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%. The lysosomal system may play some role in cell division.