The rat liver phosphoribosylpyrophosphate (PRPP) synthetase exists as complex aggregates composed of the 34-kDa catalytic subunits (PRS I and II) and other 39- and 41-kDa proteins (Kita, K., Otsuki, T., Ishizuka, T., and Tatibana, M. (1989) J. Biochem. (Tokyo) 105, 736-741), which are termed here PRPP synthetase-associated proteins (PAPs). We have cloned the cDNA for the major one of 39 kDa (PAP39) from a rat liver cDNA library. Nucleotide sequencing showed that the clone encoded 356 amino acids containing sequences of all five peptides derived from PAP39. Surprisingly, the deduced amino acid sequence is markedly similar to those of the 34-kDa catalytic subunits. Excluding two regions (about 45 residues in total), PAP39 has a 48% identity with PRS I. Northern analysis detected a major 1.9-kilobase transcript in all 16 rat tissues examined, and the relative amounts of PAP39 mRNA to PRS I mRNA varied with tissues. Covalent cross-linking experiments gave definitive evidence for molecular interaction of PAP39 with the catalytic subunits. Immunoprecipitation experiments revealed that all the catalytic subunits existed as complexes containing PAP39. When PAPs were eliminated from the rat liver enzyme complex by gel filtration in the presence of 1 m MgCl2, a lyotrope, or by mild tryptic treatment, the enzyme activity of the remaining catalytic subunits increased. Based on these results, we propose that PAP39, the major component of PAPs, plays a negative regulatory role in PRPP synthesis and hence is an important factor controlling nucleotide syntheses in general.