The effect of exposure to dexamethasone and serum-deprivation on myofibrillar protease activity was determined by following cleavage of [14C]globin by isolated myofibrils obtained from rat skeletal muscle in culture. Dexamethasone [10(-7) M] produced a 46% increase in protease activity, and serum-deprivation caused a 50% increase in activity over that of the enzyme in control cultures. The increases in proteolysis occurred concurrently with increased rate of overall protein degradation in these cells and were not associated with changes in cell viability. In cultured rat cardiac muscle cells dexamethasone failed to enhance myofibrillar protease activity, while serum-deprivation produced a 52% increase in the enzyme activity. Addition of insulin (50 mU/ml) to the cultures did not affect proteolysis or myofibrillar protease activity, but completely prevented the dexamethasone-induced increase of these activities. This effect of insulin suggests that the increase of muscle proteolysis in insulin-deficient diabetic animals reflects an enhanced response of the muscle to circulating glucocorticoids rather than a direct effect of insulin-deprivation on muscle proteolysis. Taken together, the present observations indicate that muscle cells in culture retain the ability to respond to catabolic stimuli by adaptive changes in the myofibrillar protease activity in a manner analogous to that of their parent tissue in the intact animal.