The human gene (PTGS2) encoding an inducible isozyme of prostaglandin‐endoperoxide synthase (prostaglandin‐endoperoxide synthase 2) that is distinct from the well‐characterized and constitutive isozyme (prostaglandin‐endoperoxide synthase 1), was isolated using a polymerase‐chain reaction‐generated cDNA fragment probe for human prostaglandin‐endoperoxide synthase 2. Nucleotide sequence analysis of the entire human prostaglandin‐endoperoxide‐synthase‐2 gene demonstrated that it is more than 8.3 kb in size and consists of ten exons; this gene is very similar to the murine and chicken prostaglandin‐endoperoxide‐synthase‐2 genes. The structures of exons in the human prostaglandin‐endoperoxide‐synthase‐2 gene were also similar to those of the human prostaglandin‐endoperoxide‐synthase‐1 gene (PTGS1). However, the sizes of introns in the human prostaglandin‐endoperoxide‐synthase‐2 gene were generally smaller than those of the human prostaglandin‐endoperoxide‐synthase‐1 gene. Primer‐extension analysis indicated that the transcriptional‐start site is 134 bases upstream of the translational‐initiation site. The sequence of the 1.69‐kb region of nucleotides preceding the transcriptional‐start site and the first 0.8‐kb intron contained a canonical TATA box and various transcriptional‐regulatory elements (CArG box, NF‐IL6, PEA‐1, myb, GATA‐1, xenobiotic‐response element, cAMP‐response element, NF‐κB, PEA‐3, Sp‐1 and 12‐O‐tetradecanoyl‐phorbol‐13‐acetate‐response element). The nucleotide sequence of the 5′‐flanking region (275 bp) of the human prostaglandin‐endoperoxide‐synthase‐2 gene showed 63% similarity to the sequence of murine prostaglandin‐endoperoxide‐synthase‐2/TIS10 gene, but essentially no homology to the chicken prostaglandin‐endoperoxide‐synthase‐2 gene, and human and murine prostaglandin‐endoperoxide‐synthase‐1 genes. A fluorescence in situ hybridization study showed that the human genes coding for prostaglandin‐endoperoxide synthase 1 (PTGS1) and prostaglandin‐endoperoxidase synthase 2 (PTGS2) were mapped to distinct chromosomes 9q32‐q33.3 and 1q25.2‐q25.3, respectively, indicating that these genes are not genetically linked.