The objective of this study was to determine the effects of leupeptin and two leupeptin analogs (calpain inhibitors I and II) on protein turnover in embryonic chick skeletal muscle cell cultures and on growth of chicks. For in vitro studies, inhibitors (70 μg/mL) were added to the medium of cell cultures for 24 hr and proteolysis was determined by measuring the release of natural leucine into medium containing all leucine as [5,5,5- 2H]leucine. Compared with controls, leupeptin and calpain inhibitors I and II decreased proteolysis 17.0%, 55.4%, and 60.6%, respectively. For in vivo studies, 7-day-old male White Leghorn chicks were given the inhibitors by daily intraperitoneal injection for 7 days. In experiment 1, inhibitors were administered in a 25% ethanol:75% saline carrier at doses of 2.0 or 20.0 mg/kg body weight. Calpain inhibitors I and II had no effect on lean tissue gain or feed efficiency. Leupeptin-injected chicks gained 51.1 ± 2.1 g and control chicks gained 43.2 ± 2.1 g ( P < 0.05). In experiment 2, leupeptin was tested at four levels, and in addition, a group was injected with saline only. All chicks injected with ethanol:saline grew more slowly ( P < 0.05) than the saline-injected chicks, indicating that the ethanol suppressed growth. Leupeptin increased growth over that of ethanol:saline-injected control chicks ( P < 0.05) at levels of 0.2, 2, and 20 mg/kg body weight. In experiment 3, leupeptin injected in a saline carrier was shown not to affect chick growth. These data demonstrated that, although calpain inhibitors were both three-fold more potent than leupeptin in decreasing proteolysis in vitro, they did not alter in vivo growth of chicks. Only leupeptin improved growth in vivo, and then only in chicks with growth suppressed by ethanol injection. Whether leupeptin directly affects growth or indirectly alters growth through feed intake is unknown.