Polyglutamine protein aggregation is associated with eight inherited neurodegenerative disorders. In Huntington's disease, N-terminal fragments of mutant huntingtin form intracellular aggregates and mediate cellular toxicity. Recent studies have shown that chaperones inhibit polyglutamine-mediated aggregation and cellular toxicity. Because chaperones also inhibit caspase activation to protect cells from death, it remains unclear whether the protective effect of chaperones on polyglutamine-mediated cellular toxicity is dependent on their inhibition of protein aggregation. In this study, we show that several chaperones including HSP 40, HSP 70, and N-ethylmaleimide-sensitive factor can inhibit cellular toxicity caused by N-terminal mutant huntingtin fragments. However, only HSP 40 is able to inhibit huntingtin aggregation. Furthermore, time-course study suggests that the protection of chaperones against huntingtin toxicity is not the result of their suppression of huntingtin aggregation. Chaperones inhibit caspase-3 and caspase-9 activation mediated by mutant huntingtin, and this inhibition is independent of huntingtin aggregation. We propose that the inhibition of caspase activity by chaperones is involved in their suppression of polyglutamine toxicity.