Members of the C/EBP transcription factor family regulate cell differentiation and multiple other cellular functions. The cellular levels of C/EBPalpha, gamma, delta, epsilon, and Gadd153/CHOP are regulated in part by proteasome-dependent degradation. In contrast, mechanisms regulating the degradation of C/EBPbeta are poorly understood. We tested the hypothesis that the degradation of C/EBPbeta is calpain-dependent. Studies were performed in cultured L6 myotubes (a rat skeletal muscle cell line) because we have found previously that C/EBPbeta may be involved in the regulation of muscle proteolysis. Treatment of cultured L6 myotubes with the calpain inhibitors calpeptin and Calpain Inhibitor I and II resulted in increased C/EBPbeta concentrations but did not influence cellular levels of the other C/EBP transcription factor family members. Transfection of myoblasts with a plasmid expressing the endogenous calpain inhibitor calpastatin resulted in increased cellular levels of C/EBPbeta whereas the opposite result was observed in myoblasts overexpressing micro- or m-calpain. Co-immunoprecipitation provided evidence for protein-protein interaction between C/EBPbeta and micro- and m-calpain suggesting that C/EBPbeta may be a calpain substrate. This notion was supported by experiments in which immunoprecipitated C/EBPbeta was incubated with purified micro-calpain in a cell-free system. The increase in C/EBPbeta levels caused by inhibition of calpain activity was accompanied by increased C/EBPbeta DNA-binding and gene activation. The present results suggest that C/EBPbeta is degraded by a calpain-dependent mechanism in skeletal muscle cells and that the role of calpains is specific for C/EBPbeta among different members of the C/EBP transcription factor family.