The inhibitory effect of insulin on skeletal muscle protein breakdown was tested in fed or fasted rats whose muscles had been isolated by evisceration. Degradation was estimated from the accumulation of tyrosine in the plasma after protein synthesis was blocked with cycloheximide. According to earlier results from this method, fasting for 20 hours increases proteolysis. In the present work, in fasted preparations whose plasma insulin concentrations were maintained at higher levels by intravenous infusions of insulin and glucose, proteolysis was inhibited by insulin. In fed eviscerated rats, insulin was ineffective. To examine more closely the response of fasted skeletal muscles to insulin, plasma insulin concentrations in preparations of 20-hour fasted rats were varied over a wide range by treatment with an initial bolus of insulin, and then proteolysis was measured as change in tyrosine concentration during a 90-minute period. In fasted rats, insulin inhibited proteolysis in a dose-dependent fashion, with a half-maximum concentration of 35 microU/mL. However, at insulin levels equal to concentrations found in normal fed rats, compensation for the fast-induced proteolytic effect was still incomplete. The results suggest that in these more intact preparations, insulin does have a physiologically significant inhibitory effect on fast-induced muscle protein degradation, but that changes in fasting and feeding cannot be entirely explained by the action of insulin.