Insulin receptor substrate-1 (IRS-1) is a protein expressed in 3T3-L1 adipocytes that is involved in most, if not all of the biological responses to insulin. Chronic exposure of these cells to insulin down-regulates IRS-1 by stimulating its degradation (Rice, K.M., Turnbow, M.A. and Garner, C.W. (1993) Biochem. Biophys. Res. Commun. 190, 961-967). This insulin-induced down-regulation of IRS-1 was totally abolished by BAPTA-AM (cell-permeable calcium chelator), E-64d (cell-permeable thiol protease inhibitor), Cbz-Leu-Nleu-H and Cbz-Leu-Leu-Tyr-CHN2 (selective cell-permeable calpain inhibitor peptides). Calpastatin (specific calpain inhibitor protein) also inhibited the insulin-induced down-regulation of IRS-1 in transiently permeabilized cells. In addition, 3T3-L1 adipocytes express endogenous calpain which can degrade IRS-1 in cell-free extracts. These results suggest that the insulin-induced down-regulation of IRS-1 in 3T3-L1 adipocytes is mediated by a calcium-dependent thiol protease which is sensitive to inhibition by calpain inhibitors.