Mitotic checkpoint defects of the cell cycle have been implicated in the development of human cancers. Since hBUB1 and hBUBR1, whose products function in the spindle checkpoint pathway, have been shown to be mutated in a subset of colon cancers with chromosomal instability, we investigated the contribution of these genes to lung cancer development. One hundred and two lung cancer (50 small cell lung cancers and 52 non-small cell lung cancers) and 4 mesothelioma cell line DNAs were analyzed by Southern blot analysis, but no rearrangements or deletions of hBUB1 and hBUBR1 were detected. Using single strand conformation polymorphism analysis, we studied all the 25 exons except exon 1 of the hBUB1 gene in 88 lung cancer DNAs. One lung cancer cell line, NCI-H345, showed a single nucleotide substitution, which resulted in an Arg-to-Gln change at codon 209 (CGA to CAA). Eleven cell line DNAs exhibited a single nucleotide polymorphism in intron 9 of hBUB1, all of which were heterozygous. Similar mutation analysis of hBUBR1 in 47 lung cancer cell line cDNAs revealed a frequent polymorphism at codon 349 (CAA to CGA) leading to a substitution of Gln to Arg but no mutations. Northern blot analyses showed that both hBUB1 and hBUBR1 genes were expressed in all of 31 lung cancer cell lines tested with no significant difference in the expression level. Our results suggest that alterations in hBUB1 and hBUBR1 rarely contributed to the genetic change of lung cancers.