The role of the proteasome in the regulation of cellular levels of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) is poorly understood. We tested the hypothesis that C/EBPbeta levels in cultured myotubes are regulated, at least in part, by proteasome activity. Treatment of cultured L6 myotubes, a rat skeletal muscle cell line, with the specific proteasome inhibitor beta-lactone resulted in increased nuclear levels of C/EBPbeta as determined by Western blotting and immunofluorescent detection. This effect of beta-lactone reflected inhibited degradation of C/EBPbeta. Surprisingly, the increased C/EBPbeta levels in beta-lactone-treated myotubes did not result in increased DNA-binding activity. In additional experiments, treatment of the myotubes with beta-lactone resulted in increased nuclear levels of growth arrest DNA damage/C/EBP homologous protein (Gadd153/CHOP), a dominant-negative member of the C/EBP family that can form heterodimers with other members of the C/EBP family and block DNA binding. Coimmunoprecipitation and immunofluorescent detection provided evidence that C/EBPbeta and Gadd153/CHOP interacted and colocalized in the nuclei of the beta-lactone-treated myotubes. When Gadd153/CHOP expression was downregulated by transfection of myotubes with siRNA targeting Gadd153/CHOP, C/EBPbeta DNA-binding activity was restored in beta-lactone-treated myotubes. The results suggest that C/EBPbeta is degraded by a proteasome-dependent mechanism in skeletal muscle cells and that Gadd153/CHOP can interact with C/EBPbeta and block its DNA-binding activity. The observations are important because they increase the understanding of the complex regulation of the expression and activity of C/EBPbeta in skeletal muscle.