A hallmark of cancer is inactivation of cell cycle checkpoints. However, very few mutations targeting mitotic checkpoint genes have been described, and in those instances, a wild-type copy of the gene was retained. chfr is a mitotic checkpoint gene that functions in early prophase delaying chromosome condensation in response to microtubule poisons. In a panel of 53 lung carcinomas for which matched normal tissue was available, we identified three missense mutations in the chfr gene, at least one of which was associated with loss of heterozygosity. In tissue culture checkpoint assays, the tumor-associated missense mutants had reduced activity or were inactive. Together with recent data suggesting that the chfr gene is frequently silenced in various tumors because of methylation of its promoter, these findings suggest that chfr is inactivated by multiple mechanisms in human cancer.