The rat Yoshida sarcoma (YS) cDNA library was screened using oligonucleotide probes designed from peptide sequences of rat phosphoribosylpyrophosphate synthetase, and two distinct clones were obtained. Nucleotide sequencing revealed that both clones encode 317 amino acids containing the peptide sequences. The deduced amino acid sequences of the two differ only by 13 residues (96%) identity), whereas the nucleotide sequences are relatively divergent (81% identity in the coding regions). These results, together with N-terminal amino acid sequencing data, suggest the existence of two different subunits of this enzyme, designated as PRS I and II (their genes as PRPS1 and PRPS2). Transcripts of 2.3 and 3.7 kilobases were detected in YS cells and rat liver by Northern blot analysis, using PRS I and II cDNAs respectively, as probes. In the liver, the expression of both genes increased after partial hepatectomy.