The activity of calpain, a calcium-activated protease, is required during the mitotic clonal expansion phase of 3T3-L1 embryonic preadipocyte differentiation. Here we examined the role of calpain in the adipogenesis of ST-13 preadipocytes established from adult primitive mesenchymal cells, which do not require mitotic clonal expansion. After exposure to the calpain inhibitor, N-benzyloxycarbonyl-L-leucyl-L-leucinal or overexpression of calpastatin, a specific endogenous inhibitor of calpain, ST-13 preadipocytes acquired the adipocyte phenotype. Overexpression of calpastatin in ST-13 adipocytes stimulated the expression of adipocyte-specific CCAAT/enhancer-binding protein-alpha (C/EBPalpha), peroxisome proliferator-activated receptor (PPAR)-gamma, sterol regulatory element-binding protein 1, and the insulin signaling molecules, insulin receptor alpha, insulin-receptor substrates, and GLUT4. However, insulin-stimulated glucose uptake was reduced by approximately 52%. The addition of calpain to the nuclear fraction of ST-13 adipocytes resulted in the Ca(2+)-dependent degradation of PPARgamma and C/EBPalpha but not sterol regulatory element-binding protein 1. Exposing ST-13 adipocytes to A23187 also led to losses of endogenous PPARgamma and C/EBPalpha. Under both conditions, calpain inhibitors almost completely prevented C/EBPalpha cleavage but partially blocked the decrease of PPARgamma. Two ubiquitous forms of calpain, mu- and m-calpain, localized to the cytosol and the nucleus, whereas the activated form of mu- but not m-calpain was found in the nucleus. Finally, stable dominant-negative mu-calpain transfectants showed accelerated adipogenesis and increase in the levels of PPARgamma and C/EBPalpha during adipocyte program. These results support evidence that the calpain system is involved in regulating the differentiation of adult primitive mesenchymal ST-13 preadipocytes.