We examined loss of heterozygosity (LOH) at the retinoblastoma susceptibility gene (RB1) locus on chromosome 13q14 in 20 non-small-cell lung cancers (NSCLCs) using polymorphic markers. The expression of RB protein was examined by immunohistochemical analysis of paraffin-embedded specimens of the same tumors. The results revealed that 10 of 16 informative cases showed an LOH at the RB1 locus, whereas only 2 of the 10 tumors lost expression of the RB protein. These 2 tumors had mutations in the remaining RB1 allele. Thus, inactivation of the RB1 gene appears to be involved in a small subset of NSCLCs only. To elucidate the presence of tumor-suppressor genes other than RB1 on 13q, heterozygosity at 15 different loci was investigated. Of 20 tumors analyzed, 15 showed an LOH at least at one locus, and the regions 13q12.1-qter, 13q12.2-14.2 and 13q14.1-q14.3, including the RB1 locus, were deleted in significant numbers of the tumors. Our results suggest that, in addition to the RB1 gene, abnormalities of other tumor-suppressor genes on chromosome 13q are involved in the development of human NSCLCs.