The enzymatic activity of creatine phosphokinase and the lysosomal enzymes cathepsin D and acid phosphatase was followed during skeletal muscle regeneration after partial excision to the gastrocnemius muscle in the rat. For each time interval (1, 2, 5, 14 and 45 days) following injury, the activity of the regenerated muscle was compared with the activity in the contralateral sham operated muscle. The specific activity of creatine phosphokinase of the regenerated muscle showed a significant decrease (25%) during the first 2 days post injury and thereafter was comparable to that of the uninjured control muscle. The activity of cathepsin D was 2.3-4-fold significantly higher in the regenerated muscle than in the control intact muscle from day 1 until day 14 post-injury. At 45 days after partial excision, the activity of this enzyme was comparable to a normal muscle. However, the activity of another lysosomal enzyme (acid phosphatase) did not show any distinct changes from the level of this enzyme in the uninjured muscle during the course of muscle regeneration. It is suggested that elevation of lysosomal enzymes in skeletal muscle may not be confined to conditions of muscle wasting and degradation but also to differentiation and development processes such as during muscle regeneration following injury.