Phosphoribosylpyrophosphate (PP-Rib-P) synthetase (EC 2.7.6.1) subunit I gene (PRPS1) is constitutively expressed in various tissues (Taira, M., Iizasa, T., Yamada, K., Shimada, H., and Tatibana, M. (1989) Biochim. Biophys. Acta 1007, 203-208). We report here the exon-intron organization and the transcription promoter sequence of rat PRPS1 gene. This gene has 22 kilobases and is split into 7 exons ranging in size from 99 to 251 base pairs (bp), except for exon 7 (1008 bp). A putative PP-Rib-P binding site is encoded in exon 5. The exon-intron boundaries are similar to the consensus sequences for mammalian introns. S1 nuclease and primer extension assays with the use of RNA from rat Yoshida ascites sarcoma cells led to the identification of four possible transcription start points closely spaced between 126 and 129 bp from the ATG initiation codon. In the upstream region from the transcriptional start sites, we observed a TATA-like sequence (TAATTTAAT) at nucleotides -28, a CCAAT element (AGCCAATC) at nucleotides -80, and three GC boxes (putative Sp1-binding sites) at nucleotides -103, -43, and -10. A comparison of the promoter region for PRPS1 with those of other housekeeping genes revealed a homology resembling that of the beta-actin gene.