The role of the proteasome in the regulation of cellular levels of the transcription factor C/EBP is poorly understood. We tested the hypothesis that C/EBP 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 -lactone resulted in increased nuclear levels of C/EBP as determined by Western blotting and immunofluorescent detection. This effect of -lactone reflected inhibited degradation of C/EBP. Surprisingly, the increased C/EBP levels in -lactone-treated myotubes did not result in increased DNA binding activity. In additional experiments, treatment of the myotubes with -lactone resulted in increased nuclear levels of 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. Co-immunoprecipitation and immunofluorescent detection provided evidence that C/EBP and Gadd153/CHOP interacted and co-localized in the nuclei of the -lactone-treated myotubes. When Gadd153/CHOP expression was downregulated by transfection of myotubes with siRNA targeting Gadd153/CHOP, C/EBP DNA binding activty was restored in -lactone-treated myotubes. The results suggest that C/EBP is degraded by a proteasome-dependent mechanism in skeletal muscle cells and that Gadd153/CHOP can interact with C/EBP 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/EBP in skeletal muscle.