Most human cancers involve multiple genetic changes, including activation of oncogenes such as Ki-ras-2 (Kras2) and inactivation of any one of a number of tumor suppressor genes such as p53 and members of the retinoblastoma (Rb) regulatory axis. As part of an ongoing project to determine how in utero exposure to chemical carcinogens affects the molecular pathogenesis of murine lung tumors, the p53 and p16 Cdkn2a genes were analyzed by using paraffin-embedded lung tissues from mice treated transplacentally with 3-methylcholanthrene. Single-strand conformation polymorphism analysis of exons 5-8 of the p53 gene, as well as their flanking introns, demonstrated an absence of mutations at this gene locus. However, a genetic polymorphism was identified at nt 708 in intron 4 of the DBA/2 strain of mice 5 bp downstream of a 3Β΄ branching-point splice signal. Analysis of exons 1 and 2 of the Cdkn2a gene by single-strand conformation polymorphism and sequence analyses revealed mutations in exon 2 in 7% of the tumors examined. Tumor 23-1 exhibited a CACβTAC transition at nt 301 (His 74 βTyr 74 ), and tumor 36-1 exhibited a GGGβGAG transition at nucleotide 350 (Gly 90 βGlu 90 ). Northern blot analysis of 14 of the larger tumors showed a marked decrease in the levels of Rb RNA expression. Immunohistochemical analysis revealed a spectrum of pRb expression, with the smaller adenomas showing moderate numbers of nuclei with heterogeneous staining for pRb in contrast with a highly reduced or near-complete absence of expression in the nuclei of larger tumors with features of adenocarcinomas. The low incidence of mutations at tumor suppressor loci suggested that inactivation of tumor suppressor genes was a late event in murine lung tumor pathogenesis. The identification of both mutations at the Cdkn2a gene locus and reduced levels of Rb expression combined with previous studies demonstrating a high incidence of mutated Kras2 alleles in these tumors implies that alterations of the Rb regulatory axis, in combination with mutation of Kras2, may be the preferred pathway for the pathogenesis of pulmonary tumors in transplacentally exposed mice.