Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG expansion that results in elongation of the polyglutamine tract at the N terminus of huntingtin (Htt). Abnormal proteolytic processing of mutant Htt has been implicated as a critical step in the initiation of HD. The protease(s) involved in this process has not been fully characterized. Here we report that activated calpain was detected in the caudate of human HD tissue but not in age-matched controls. In addition, one of the major N-terminal Htt proteolytic fragments found in human HD tissue appears to be derived from calpain cleavage. Htt fragments in HD lysates were similar in size to those produced by exposure of in vitro-translated Htt to exogenous calpain. Incubation of in vitro-translated Htt with calpain generated a cascade of cleavage events with an initial intermediate cleavage product at 72 kDa and a final cleavage product at 47 kDa. The rate of cleavage of Htt by calpain was polyglutamine-length-dependent. These results suggest that cleavage of Htt in human HD tissue is mediated in part by the Ca²⁺-activated neutral protease, calpain.