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Cultured L6 myoblasts afford considerable advantages for identifying and studying the insulin-like actions of test substances in a muscle-derived line. We have used this system to examine the interaction of the oral hypoglycemic sulfonylurea glyburide with bovine insulin on protein degradation and synthesis as well as on thymidine incorporation (as a measure of DNA synthesis) in these cells. Bovine insulin, at doses of 0.1 μg/mL to 10 μg/mL, produced a dose-dependent inhibition of protein degradation (measured by release of trichloracetic acid (TCA)-soluble 14C-tyrosine from myoblasts into the culture medium) and increase in total protein content in the cultured myoblasts. At concentrations of 10 μg/mL, insulin achieved its maximal suppression of protein degradation (by nearly 50%) and increased cellular protein content (by 15%) over levels observed in the absence of added insulin. Glyburide, at concentrations at or above 1 μg/mL, significantly suppressed protein degradation (up to 14%) and slightly augmented protein content of the cells. The effects of glyburide on protein degradation were additive with those of submaximally but not maximally effective concentrations of insulin, suggesting a common mechanism of action of the compounds. Both insulin and glyburide, at maximally effective doses, significantly depressed protein degradation as early as 2 to 6 hours after exposure. In addition, in a 24-hour labeling experiment, insulin stimulated tyrosine incorporation into TCA-insoluble protein and thymidine incorporation into DNA in the cells, whereas glyburide did not enhance these processes and, under certain conditions, inhibited them. These results demonstrate that glyburide, either alone or in concert with insulin, is capable of significantly inhibiting protein turnover in skeletal muscle-derived cells. The physiological implications of these observations are discussed.