A 600-base pair cDNA containing the entire rat N-ras coding exonic sequence was amplified by a transcript-PCR method. Sequence comparison of mammalian N-ras genes revealed that the rat N-ras proto-oncogene shared extensive sequence homology with human and mouse N-ras proto-oncogenes. The homology of N-ras in human, mouse, and rat was 90 to 95% in nucleotide sequence. Rat N-ras exhibited 4 amino acid differences when compared to mouse N-ras, 3 of which were in the C-terminal region encoded by exon IV. An evolutionary divergence at position 69 was also found, in which rat N-ras contained glycine but other known mammalian ras proteins including H-, K-, and N-ras have aspartate at position 69 in this highly conserved region. Starting from total RNA derived from the liver of Fischer rat, a double strand cDNA containing two unique endonuclease sites in its termini was obtained within 5 hrs. The terminal endonuclease sites were used for direct cloning into M13 to obtain a library enriched for N-ras proto-oncogene. Screening assay of plaques and sequence analysis of individual cloned cDNA product were used to evaluate the efficency and fidelity of this cDNA amplification-direct M13 cloning procedure. The target N-ras cDNA clones were detected in 5% of the total screened plaques and no sequence errors introduced by the in vitro amplification procedure were observed. This method proved satisfactory for obtaining a complete cDNA sequence of the N-ras proto-oncogene from nanogram quantities of the total liver RNA.