Phosphoribosylpyrophosphate (PRPP) synthetase is a key enzyme for de novo and salvage syntheses of nucleotides. PRPP synthetase activity is distributed in almost all tissues. cDNA cloning of rat PRPP synthetase showed the existence of two distinct types of subunits, referred to as PRS I and II (JBC 262; 14867, ′87). Northern blot analysis of RNAs from various mammalian tissues, using rat PRS I and PRS II cDNA as probes, showed that the expression of each gene is regulated in a tissue-specific manner: mRNAs of rat PRS I and/or PRS II were highly detected in brain, adrenal gland, spleen, lung, thymus, adipose tissue, and testis. The sizes of mRNAs for PRS I and II were 2.3 kb and 3.7 kb (2.7 kb for humans), respectively. In addition, in testes of rat, mouse, and humans, a unique PRS I-related 1.4 kb transcript was found, together with regular PRS I and II mRNAs. To characterize human PRS I and II mRNA as well as testis-specific one, the cDNA library was constructed in lambda phage vector λgt 10 from human testicular poly(A)+RNA and 1.35 × 106 recombinants were screened with rat PRS I and II cDNAs as probes. Two types of clones were obtained: with PRS I cDNA probe, clones with a 1.2 or 1.1 kb insert and with PRS II probe, clones with a 2.4, 2.3, 2.1, or 1.8 kb insert. The former group covered the full coding region and may correspond to the specific 1.4 kb transcript and the latter to the PRS II transcript.