The activity of the thiolproteases, cathepsins B, H, and B + L, one of the most important groups of endoproteases, was measured in skeletal and myocardial muscle and liver of Sprague-Dawley rats submitted to fasts of different duration (control and 24, 48, and 72 h). After the fasting period, the animals were killed, and fresh tissue samples were collected. Enzyme activity was determined in vitro with the specific substrates Z-Arg-Arg-MCA for cathepsin B, Z-Phe-Arg-MCA for cathepsin B + L, and Arg-MCA for cathepsin H. Results show different patterns in the organs studied: activity increased linearly in liver, decreased in myocardial muscle, and had no change in skeletal muscle. These results suggest that the expected alteration observed in proteolytic activity in fasted tissues is produced to a certain degree by changes in thiolprotease activity.