We have detected nucleotide sequences related to the transforming gene of avian sarcoma vius (ASV) in the DNA of uninfected vertebrates. Purified radioactive DNA (cDNAsarc) complementary to most of all of the gene (src) required for transformation of fibroblasts by ASV was annealed with DNA from a variety of normal species. Under conditions that facilitate pairing of partially matched nucleotide sequences (1.5 M NaCl, 59 degrees), cDNAsarc formed duplexes with chicken, human, calf, mouse, and salmon DNA but not with DNA from sea urchin, Drosophila, or Escherichia coli. The kinetics of duplex formation indicated that cDNAsarc was reacting with nucleotide sequences present in a single copy or at most a few copies per cell. In contrast to the preceding findings, nucleotide sequences complementary to the remainder of the ASV genome were observed only in chicken DNA. Thermal denaturation studies of the duplexes formed with cDNAsarc indicated a high degree of conservation of the nucleotide sequences related to src in vertebrate DNAs; the reductions in melting temperature suggested about 3--4% mismatching of cDNAsarc with chicken DNA and 8--10% mismatching of cDNAsarc with the other vertebrate DNAs.