The gene for the murine prostaglandin endoperoxide (PGH) synthase (8, 11, 14-eicosatrienoate, hydrogen-donor:oxygen oxidoreductase, EC 220.127.116.11) has been cloned. The gene was isolated from a mouse NIH 3T3 cell genomic library and is contained in four overlapping lambda FIXII bacteriophage clones. The gene spans approximately 22 kb and consists of 11 exons. Primer extension and RNAse protection assays indicate that transcription of the gene begins at an initiation site 63 nucleotides 5' to the ATG translation initiation codon. Neither TATA or CAAT boxes are present immediately upstream of the transcriptional start site, but SP1 binding sites are present at positions -47 to -42 and -30 to -25, relative to the transcription initiation site. Examination of the 5'-end and 2400 bp of the 5'-flanking sequence of the gene revealed sequences with homology to several transcriptional regulatory sequences. Three putative AP-1 binding sites were found, two within the first exon and intron and another at position -2097 to -2090. The AP-1 site at position -2097 is adjacent to a sequence with similarity to a negative glucocorticoid regulatory element (nGRE) (position -2123 to -2009). The presence of AP sites by themselves, or in conjunction with an nGRE sequence, suggests a possible interplay between jun/fos regulatory proteins and the glucocorticoid receptor for positive and negative regulation of the PGH synthase gene. An unexpected finding was the presence at position -403 to -385 of a putative dioxin responsive element, a sequence found to be responsible for the induction of transcription of the cytochrome P450IA1 gene (CYPIA1) and other genes involved in detoxification/activation of polycyclic aromatic hydrocarbons.