Because protein degradation in liver and skeletal muscle is increased by thyroid hormones and decreased by thyroidectomy; we investigated the influence of thyroid hormones on the level of lysosomal enzymes. Hypophysectomized rats received daily injections of L-thyroxine or L-triiodothyronine. After 3 days of this regimen, homogenates of liver and skeletal muscle showed a 2- to 3-fold increase in the activities of cathepsin D, cathepsin B, and other lysosomal enzymes including leucine aminopeptidase, acid phosphatase, β-galactosidase, N-acetylglucosaminidase, and α-mannosidase. In liver, this effect reflected increased enzyme activity in the two subcellular fractions that normally contain lysosomes. Titration of cathepsin D with pepstatin indicated that the increase in this activity resulted from an increase in the number of enzyme molecules. These effects occurred with both pharmacologic (thyrotoxic) and physiologic (growth-promoting) doses of thyroid hormones. Liver and skeletal muscle from thyroidectomized rats had approximately 50% of the normal levels of lysosomal enzyme activities. Under these various conditions, heart and kidney, tissues in which protein degradation does not appear to be influenced by thyroid hormones, showed no significant changes in lysosomal hydrolases. Thus, thyroid hormones regulate proteolytic and other lysosomal enzyme activities in those tissues in which these hormones influence protein degradation. Many characteristic features of hyperthyroidism and hypothyroidism may result from changes in levels of lysosomal enzymes.