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 (UOsm) was similar in groups of EP3 −/− and wild-type (EP3 +/+) mice. However, after inhibition of endogenous PGE2 production by indomethacin, UOsm 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.