We have cloned, by the polymerase chain reaction (PCR), two rat genomic fragments of 1.3 and 1.2 kb, both of which hybridize to a human p53 cDNA probe. Nucleotide sequencing revealed that they are two intronless rat p53 pseudogenes (designated as psi R53-1 and psi R53-2, respectively), representing a start-to-stop-codon-length copy of the processed transcript of the rat p53 gene. Further PCR analysis of DNA from rat organs (skin, kidney, etc.) of two different strains demonstrated that psi R53-1 and psi R53-2 were formed in the germ-line. As psi R53-1 and psi R53-2 share 85 and 83% homology with the rat p53 cDNA, it is thus estimated that psi R53-1 was created approx. 10 million years (Myr) ago and psi R53-2 arose 12 Myr ago. Moreover, GenBank scanning indicated that a 95-bp insert in psi R53-1, as compared with the cDNA, was 90% homologous with a sequence of mouse alternatively spliced p53 mRNA, where the spliced p53 mRNA contains an additional 96 nt derived from intron 10. Although the rat alternatively spliced p53 mRNA has so far not been described, our data suggest that these two processed pseudogenes may have been generated by integration of different mRNA intermediates into germ-line DNA.