Prostamide/prostaglandin F synthase (PM/PGFS) is an enzyme with very narrow substrate specificity and is dedicated to the biosynthesis of prostamide and prostaglandin F2α. The importance of this enzyme, pg. 2 relative to the aldoketoreductase (AKR) series, in providing functional tissue prostamide F2α levels was determined by creating a line of PM/PGFS gene deleted mice. Deletion of the gene encoding PM/PGFS (fam213b) was accomplished by a two exon disruption. Prostamide F2α levels in WT and PM/PGFS KO mice were determined by LC/MS/MS. Intraocular pressure was measured by tonometry and outflow facility was measured by the iPerfusion method in enucleated mouse eyes. Deletion of fam213b had no observed effect on behavior, appetite, or fertility. Intraocular pressure was significantly elevated by approximately 4 mmHg in PM/PGFS KO mice compared to littermate WT mice. No effect on pressure dependent outflow facility occurred, which is consistent with the typically reported prostanoid effect of increasing outflow through the unconventional pathway. The elevation of intraocular pressure caused by deletion of the gene encoding the PM/PGFS enzyme likely results from a diversion of the endoperoxide precursor pathway to provide increased levels of those prostanoids that raise intraocular pressure, namely prostaglandin D2 (PGD2), thromboxane A2 (TxA2). It follows that PM/PGFS may serve an important regulatory role in the eye by providing PGF2α and prostamide F2α to constrain the influence of those prostanoids that raise intraocular pressure. Keywords(a) Expertise key words: prostaglandins, eye, animal models, enzymology, gene expression (b) Free-form key words prostamide/prostaglandin F synthase, fam213b, prostamide F2α, prostaglandin F2α, , intraocular pressure, 13). More recently, a much more substrate specific enzyme and a member of the thioredoxin-like superfamily was discovered and was designated prostamide/prostaglandin F synthase (14,15), PM/PGFS, fam213. This enzyme facilitates a more restricted and targeted formation of PGF2α and prostamide F2α and thereby conferring tighter control of regulatory function(s). Gene deletion is a classical approach for discovering biological functions and the first results of these studies are presented herein, together with the methodology for creating prostamide/prostaglandin F synthase (fam213) knock-out mice. Given the importance of PGF2α and prostamide F2α in the treatment of glaucoma, these present studies focused on regulation of intraocular pressure. pg. 4Methods Creation of prostamide/prostaglandin F synthase (fam213) knock-out mice.The prostamide/prostaglandin F synthase (PM/PGFS) knock-out mouse was generated using the RENKA embryonic stem (ES) cell line, derived from C57BL/6N mice (16). To construct a PM/PGFS targeting vector, a 990 bp DNA fragment carrying exons 2 and 3, containing the enzyme active site of PM/PGFS, was amplified by PCR, and inserted into the targeting vector as described previously (17). In this clone, a DNA fragment of a PGK promoter-driven neo-poly (A...