Aneuploidy is frequently observed in many types of human cancer cells, suggesting that mutations of genes required for chromosomal stability may occur in human tumors. The BUB gene is a component of the mitotic checkpoint in budding yeast that delays anaphase in the presence of spindle damage thus increasing the probability of successful delivery of a euploid genome to each daughter cell. Recently, human homologues of the BUB gene were identified and mutant alleles of hBUB1 were detected in two colorectal tumor cell lines. Transfection of one mutant allele led to dominant disruption of the mitotic checkpoint control in a euploid cell, suggesting that aneuploidy in some tumors could be due to defects in the mitotic checkpoint. We analyzed the entire coding sequence of hBUB1 for mutation in 31 head and neck squamous cell carcinoma (HNSCC) and lung cancer cell lines, most with severe aneuploidy. We found expression of the hBUB1 gene in all cell lines and only a single nucleotide substitution in one cell line without a resultant change in amino acid sequence. Our study demonstrates that hBUB1 is rarely a target for genetic alterations in tumors of the respiratory tract.