The rat fibroblast cell line Rat 1 was transfected with total DNA of a gastrocarcinoma cell line, BGC-823. The transforming gene was cloned from the genomic library of the secondary transformants using in situ hybridization with a probe of the human Alu repeat sequence. This cloned gene is homologous to the protooncogene c-Ha-ras. The activation lesion of the transforming gene was identified by sequence analysis as a single nucleotide substitution of thymine for guanine in the 12th codon. This results in the substitution of valine for glycine at the 12th amino acid of the Mr 21,000 protein.