PRPP synthetase from rat liver exists as large molecular weight aggregates composed of at least three different components. Cloning of cDNA for the catalytic subunit revealed the presence of two highly homologous isoforms of 34 kDa, designated as PRS I and PRS II. Northern blot analysis showed tissue-differential expression of the two isoform genes. cDNA was expressed in E. coli and studies on the recombinant isoforms showed differences in sensitivity to inhibition by ADP and GDP and to heat inactivation. The rat gene for PRS I has 22 kb and is split into 7 exons. cDNAs for human enzymes were also cloned. Human genes for PRS I and PRS II are localized at different regions on the X-chromosome and their promoter regions were examined. Another component, PRPP synthetase-associated protein of 39 kDa (PAP39), was cloned from cDNA library of the rat liver. The deduced amino acid sequence of PAP39 is remarkably similar to those of PRS I and PRS II. Evidence indicated molecular interaction between PAP39 and the catalytic subunits and an inhibitory effect of PAP39 on the catalytic activity. Expression of the PAP39 gene is tissue-differential like the PRS genes, indicating that the composition of PRPP synthetase may differ with the tissue, hence properties of the enzyme would differ. Further studies on these components and their interaction are expected to reveal various mechanisms governing mammalian PRPP synthetase.