Livers of a natural population of winter flounder from a contaminated site in Boston Harbor were examined for the presence of oncogenes by transfection of DNA into NIH3T3 mouse fibroblasts. Tissues analyzed contained histopathologic lesions including abnormal vacuolation, biliary proliferation, and, in many cases, hepatocellular and cholangiocellular carcinomas. Fibroblasts transfected with liver DNA samples from 7 of 13 animals were effective in the induction of subcutaneous sarcomas in nude mice. Further analysis revealed the presence of flounder c-K-ras oncogenes in all subcutaneous tumors examined. Direct DNA sequencing and allele-specific oligonucleotide hybridization following polymerase chain reaction DNA amplification of the tumor DNA showed mutations in the 12th codon in this gene. Analysis of DNA of all nude mouse tumors as well as all 13 diseased flounder livers showed mutations at this codon. Liver DNA samples from 5 histologically normal livers of animals from a less polluted site were ineffective in the transfection assay and contained only wild-type DNA sequences. The prevalence of mutations in this gene region correlated with the presence of liver lesions and could signify DNA damage resulting from environmental chemical exposure.