Machado-Joseph disease (MJD) is an inherited neurodegenerative disorder caused by the expansion of the polyglutamine stretch in the MJD gene-encoded protein, ataxin-3. Using a series of deletion constructs expressing ataxin-3 fragments with expanded polyglutamine stretches, we observed aggregate formation and cell death in cultured BHK-21 cells. The cytotoxic effect of N-terminal-truncated ataxin-3 with the expanded polyglutamine tract was enhanced under serum starvation culture, in which cells were arrested in the G(0)/G(1)phase. Coexpression of p21 (waf1/cip1/sdi1), a cyclin-Cdk inhibitor that induced cell cycle arrest in the G(1)phase, also increased the cell death susceptibility produced by the mutant ataxin-3 fragment in BHK-21 cells. The elevated susceptibility to cell death in the G(0)/G(1)phase was confirmed in nerve growth factor-treated, postmitotic neuronal PC12 cells compared with undifferentiated proliferating PC12 cells. These results strongly suggest that the cellular toxicity of truncated ataxin-3 with an expanded polyglutamine stretch is enhanced by cell cycle arrest in the G(0)/G(1)phase. Mutant ataxin-3 may confer a higher susceptibility to cell death on cells in the G(0)/G(1)phase.