Organic anion transporter 6 (Oat6; Slc22a20), a member of the OAT family, was demonstrated previously to mediate the transport of organic anions (Am J Physiol Renal Physiol 291:F314-F321, 2006). In the present study, we sought to further delineate the function of murine Oat6 (mOat6) by analyzing the effect of select organic anions on mOat6-mediated transport by using a Chinese hamster ovary (CHO) cell line stably expressing mOat6 (CHO-mOat6). When examined, kinetic analysis demonstrated that the mechanism of inhibition of mOat6 and mOat3 was competitive. Homovanillic acid, 5-hydroxyindole acetic acid, 2,4-dihydroxyphenylacetate, hippurate, and dehydroepiandrosterone sulfate (DHEAS) each significantly reduced mOat6 activity with inhibitory constant (K(i)) values of 3.0 +/- 0.5, 48.9 +/- 10.3, 61.4 +/- 7.1, 59.9 +/- 4.9, and 38.8 +/- 3.1 microM, respectively. Comparison to K(i) values determined for mOat3 (67.8 +/- 7.2, 134.5 +/- 27.0, 346.7 +/- 97.9, 79.3 +/- 4.0, and 3.8 +/- 1.1 microM, respectively) revealed that there are significant differences in compound affinity between each transporter. Fluoroquinolone antimicrobials and reduced folates were without effect on mOat6-mediated uptake. Investigation of testicular cell type-specific expression of mOat6 by laser capture microdissection and quantitative polymerase chain reaction revealed significant mRNA expression in Sertoli cells, but not in Leydig cells or spermatids. Overall, these data should aid further refinements to the interpretation and modeling of the in vivo disposition of OAT substrates. Specifically, expression in Sertoli cells suggests Oat6 may be an important determinant of blood-testis barrier function, with Oat6-mediated transport of estrone sulfate and DHEAS possibly representing a critical step in the maintenance of testicular steroidogenesis.