Recently, we have found two major physiological forms of retinoid X receptor alpha (RXR alpha): the mature 54 kDa RXR alpha and the truncated 44 kDa RXR alpha lacking a portion of N-terminal A/B domain in human and rodent livers. In this communication, we show that m-calpain was active to digest 54 kDa RXR alpha in the human hepatoma-derived cell line, HuH7, nuclei to 44 kDa fragment through 47 kDa intermediate in vitro. Although both proteolytic fragments were revealed by anti-RXR alpha antibody against its E-domain, neither fragment reacted with anti-RXR alpha antibody specific for A/B domain. The profile of the calpain-induced proteolytic fragmentation of RXR alpha was almost identical to that endogenous RXR alpha in nonmalignant human and normal mouse liver nuclei. This is the first demonstration that of RXR alpha is a substrate for m-calpain, strongly suggesting that the enzyme might also be involved in post-translational modification of the receptor in hepatocytes.