The profound influence of glucocorticoid hormones on the inflammatory response includes a rapid and significant reduction in the synthesis of cyclooxygenase (prostaglandin G/H synthase, PGHS), the key enzyme for prostaglandin biosynthesis. In analyzing the glucocorticoid effects on PGHS synthesis in C127 mouse fibroblasts, we detected a novel 4-kilobase (kb) mRNA that is related to a PGHS cDNA cloned from an ovine seminal vesicle library. This RNA is much more prevalent in cycloheximide-treated cells and, based on stringency analysis and preliminary sequence data, arises from a gene distinct from that transcribed into the previously cloned 2.8-kb PGHS cDNA. Furthermore, the 4-kb mRNA encodes a 70-kDa protein that is specifically immunoprecipitated by anti-PGHS serum. The abundance of the 4-kb mRNA is strongly decreased by dexamethasone and increased by serum within 2 h whereas the 2.8-kb PGHS mRNA, which is also seen in these cells, does not consistently change. These changes in the level of the 4-kb mRNA with serum and dexamethasone treatment parallel changes in the level of synthesized PGHS protein detected in both metabolically labeled cells and in in vitro translated mRNAs. This discovery of a cyclooxygenase-related gene that is transcriptionally regulated by serum and glucocorticoid hormones in a manner identical to that reported for cyclooxygenase activity may help clarify issues regarding cyclooxygenase regulation and suggests that two distinct and differentially regulated cyclooxygenase species exist.