A 3-kilobase-pair gene for rat brain prostaglandin D synthase [(5Z,13E)-(15S)-9 alpha,11 alpha-epidoxy-15-hydroxyprosta-5,13- dienoate D-isomerase, EC 18.104.22.168], which belongs to the lipocalin family, was isolated from a rat genomic DNA library by plaque hybridization with the cDNA for the enzyme. The gene contains seven exons, and all the splice donor and acceptor sites conform to the GT/AG rule. Transcription initiates at a guanine residue 39 base pairs upstream of the translation initiation codon, as determined by primer-extension analysis of rat brain mRNA. The 5'-flanking region of the gene lacks typical transcriptional regulatory sequences, such as TATA and CAAT boxes, but contains several sets of inverted repeats, direct repeats, and sequences resembling the transcriptional factor Sp1-binding site. The gene structure of prostaglandin D synthase is remarkably analogous to those of other lipocalins, such as beta-lactoglobulin, alpha 2-urinary globulin, placental protein 14, and alpha 1-microglobulin, in terms of number and sizes of exons and phase of splicing of introns. Furthermore, in a multiple alignment of the deduced amino acid sequences, positions of exon/intron junction of the prostaglandin D synthase gene are highly conserved and located around the positions of those of the genes for other lipocalins despite a weak homology.