The renal reabsorption and/or excretion of organic anions is mediated by specific transporters (OATs). OAT2 (Slc22a7) has been identified in rat kidney, where its mRNA expression exhibits gender differences (females (F)>males (M)). The exact localization of OAT2 protein in kidney is unknown. We studied the expression of OAT2 mRNA by RT-PCR and its protein by immunoblotting (WB) and immunocytochemistry (IC) in kidneys of adult intact and gonadectomized M and F, sex hormone-treated castrated M, and prepubertal M and F rats, and the protein in adult mice. In adult rats, the expression of OAT2 mRNA was predominant in the outer stripe (OS), exhibiting gender dependency (F>M), upregulation by castration and downregulation by ovariectomy, as well as strong downregulation by testosterone-, and weak upregulation by estradiol- and progesterone-treatment. An antibody against the rat OAT2 on WB of isolated membranes labeled a ~66 kDa protein band, that was stronger in F. By IC, stained was brush-border (BB) of the proximal tubule S3 segment (S3) in the OS and medullary rays (F>M). In variously treated rats, the pattern of 66 kDa band density in isolated membranes, and the staining intensity of BB in S3 matched the mRNA expression. The expression of OAT2 protein in prepubertal rats was low and gender-independent. In mice, the expression pattern resembled that in rats. Therefore, OAT2 in rat (and mouse) kidney is localized to the BB of S3, exhibiting gender differences (F>M) that appear in puberty and are caused by strong androgen inhibition and weak estrogen and progesterone stimulation.