Epoxyeicosatrienoic acids (EETs) confer vasoactive and cardioprotective functions. Genetic analysis of the contributions of these short-lived mediators to pathophysiology has been confounded to date by the allelic expansion in rodents of the portion of the genome syntenic to human CYP2J2, a gene encoding one of the principle cytochrome P450 epoxygenases responsible for the formation of EETs in humans. Mice have eight potentially functional genes that could direct the synthesis of epoxygenases with properties similar to those of CYP2J2. As an initial step towards understanding the role of the murine Cyp2j locus, we have created mice bearing a 626-kb deletion spanning the entire region syntenic to CYP2J2, using a combination of homologous and site-directed recombination strategies. A mouse strain in which the locus deletion was complemented by transgenic delivery of BAC sequences encoding human CYP2J2 was also created. Systemic and pulmonary hemodynamic measurements did not differ in wild-type, null, and complemented mice at baseline. However, hypoxic pulmonary vasoconstriction (HPV) during left mainstem bronchus occlusion was impaired and associated with reduced systemic oxygenation in null mice, but not in null mice bearing the human transgene. Administration of an epoxygenase inhibitor to wild-type mice also impaired HPV. These findings demonstrate that Cyp2j gene products regulate the pulmonary vascular response to hypoxia.