A stable isotope labelling method was developed for use in identifying compounds that affect the rate of proteolysis in primary embryonic chick skeletal muscle cell cultures. Effects of leupeptin and calpain inhibitors I and II on proteolysis in 8 d-old cell cultures was determined by prelabelling cells with natural leucine and measuring its release into culture medium containing [superscript]2H[subscript]3-leucine. Compared with controls, leupeptin decreased proteolysis by 6-20% (P < 0.05) and no dose effect was seen (46 to 93 [mu]g/ml). Calpain inhibitor I decreased proteolysis by 45% at 7 [mu]g/ml (P < 0.05) and further by 55-63% at the 23, 70, and 93 [mu]g/ml levels (P < 0.05). Calpain inhibitor II exhibited a significant (P < 0.05) response to dose by decreasing proteolysis from 11-61% from 7 to 70 [mu]g/ml, with no further decrease at 93 [mu]g/ml;Cycloheximide was used to block protein synthesis in muscle cultures to ascertain if the relative rates of proteolysis measured reflected absolute rates. All stable isotope labelling was used with [superscript]13C-leucine labelling on d 2-7 and natural leucine labelling on d 8. A decrease in proteolysis in response to cycloheximide was seen in contrast to the increase expected if cycloheximide had blocked synthesis with no effect on proteolysis. This invalidates the use of cycloheximide to obtain absolute rates of proteolysis;Lastly the affect of leupeptin and calpain inhibitors I and II on the growth of chicks was examined. 7-day-old male White Leghorn chicks were given daily intraperitoneal inhibitor injections for 7 days. In experiments 1 and 2, 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 gain or feed efficiency, while leupeptin-injected chicks gained more than the control chicks (P < 0.05). In experiment 2, leupeptin at four levels and a saline-injected group were tested. All chicks injected with ethanol/saline grew less (P < 0.05) than the saline-injected chicks, indicating the ethanol suppressed growth. Leupeptin increased growth over that of ethanol/saline injected control chicks (P < 0.05) at 0.2, 2, and 20 mg/kg body weight. In experiment 3, leupeptin injected in saline was shown not to affect chick growth.