The effects of sex hormones on the level of rat organic anion transporter 3 (rOAT3) protein and its localization were studied in rat kidney. Western blot analysis detected both minor 100-kDa and major 69-kDa proteins in the crude plasma membrane fraction of the kidney. The sum of the levels of both proteins in male rats was not different from that in castrated rats, testosterone-treated rats that had been castrated or female rats. In ovariectomized female rats, the level of these proteins was 3-4 times higher than that in other groups. Treatment of ovariectomized female rats with estradiol reduced rOAT3 to a normal level. Immunohistochemical analysis indicated that rOAT3 was expressed in the renal cortex and outer medulla, where this protein localized in the basolateral membrane of tubules. The immunological localization of rOAT3 was similar in the 6 experimental groups. These results suggest that the cellular level of rOAT3 protein is regulated, at least in part, by estradiol in rat kidney.