The actions of prostaglandin (PG) E₂ are mediated by four distinct classes of PGE₂E-prostanoid (EP) receptors (EP₁through EP₄). However, the in vivo functions of the individual EP receptor subtypes have not been delineated. To study the functions of one of these subtypes, the EP₃ receptor, we generated EP₃-deficient (−/−) mice by gene targeting. EP₃ −/− animals survived in expected numbers, reproduced, and had no obvious abnormalities in their major organ systems. Because the EP₃ receptor is expressed at high levels in the renal medulla and cortical collecting duct, and because previous studies have suggested that the EP₃ receptor might antagonize the effects of vasopressin in the distal nephron, we examined urinary concentrating functions in EP₃−/− mice. Basal urine osmolality (U_Osm) was similar in groups of EP₃ −/− and wild-type (EP₃ +/+) mice. However, after inhibition of endogenous PGE₂ production by indomethacin, U_Osm increased significantly in EP₃ +/+ but not in EP₃ −/− mice. Despite this insensitivity to acute inhibition of prostanoid production, EP₃ −/− mice concentrated and diluted their urine normally in response to a series of physiological stimuli. This suggests that PGE₂ acts through the EP₃ receptor to modulate urinary concentrating mechanisms in the kidney, but these effects are not essential for normal regulation of urinary osmolality.