The DNA of 22 fibrosarcomas, newly induced in BALB/c mice by subcutaneous doses of 3-methylcholanthrene (3-MCA), was tested in NIH 3T3 transformation assay. Activation of K-ras and N-ras was found in 7 and 3 cases respectively. No H-ras activation was detected. Polymerase chain reaction and oligonucleotide hybridization performed on the DNA of the 22 sarcomas revealed 5 cases of K-ras mutation at codon 12, 3 at codon 13 and 1 at both codons. One case of K13 mutation was not detectable by transfection. Three cases of mutation at codon 61 of N-ras were also found, one of which was simultaneous with a K12 mutation. Tumor-specific transplantation antigens (TSTA) were assessed in the 22 original tumors. Altogether 16 sarcomas were immunogenic, with the highest frequency of TSTA+ tumors (10/11 and 5/6) in the groups given 1.0 and 0.1 mg of 3-MCA respectively, the lowest (1/5) in that with 0.01 mg of carcinogen; ras mutations occurred in the DNAs of 11 out of the 16 TSTA+ sarcomas, but none of the DNAs of the 6 TSTA- tumors showed ras mutation. The results suggest that 3-MCA-induced transformation of subcutaneous fibroblasts can involve mutations in codons 12, 13 or 61 of K- and N- but not H-ras gene and that such mutation is accompanied by the expression of TSTA.