Alterations in multiple phosphorylation sites on exon 3 of the β‐catenin gene have recently been implicated in hepatocarcinogenesis in humans as well as mice. To identify genetic alterations which could be involved in the chemical‐induced hepatocarcinogenesis of rats, we analyzed the status of the sites in the β‐catenin gene (Ctnnb1) of liver neoplasms induced by diethylnitrosamine (DEN) in male F344 rats, using the polymerase chain reaction‐single strand conformation polymorphism method. In the present investigation, we examined 35 hepatocellular neoplasms (28 adenomas and 7 carcinomas) for the expression of mutations in the region of the β‐catenin gene. Point mutation at codon 32, 35, 37 or 41, which has been reported in human and mouse liver cell carcinomas and/or other cancers, was recognized in eleven (31%) out of 35 lesions (8 adenomas and 3 carcinomas). Our results indicate that Ctnnb1 mutations may contribute to hepatocarcinogenesis in rats. Our finding that Ctnnb1 mutation was present in adenomas as well as carcinomas also suggests that the mutation is a relatively early event in DEN‐induced hepatocarcinogenesis in rats.