Myo-inositol is a compatible osmolyte used by cells which are challenged by variations in extracellular osmolarity, as in the renal medulla. In order to accumulate large quantities of this polyol, cells rely on Na+ dependent transporters such as SMIT1. We have recently identified a second Na+/myo-inositol cotransporter, SMIT2, which presents transport characteristics corresponding to those recently described for the apical membrane of renal proximal tubules. In order to further characterize this transport system, we transfected MDCK cells with rabbit SMIT2 cDNA and selected a stable clone with a high expression level. The accumulation of radiolabelled myo- inositol by this cell line is twenty-fold larger than that seen in native MDCK cells. The affinity for myo- inositol of MDCK cells transfected with SMIT2 is slightly lower (Km = 334 µM) than that found in voltage- clamped Xenopus laevis oocytes expressing SMIT2 (Km = 120 µM). Transport studies performed using semi- permeable filters showed fully apical targeting of the SMIT2 transporter. This apical localization of SMIT2 was confirmed by transport studies on purified rabbit renal brush border membrane vesicles (BBMV). Using a purified antibody against SMIT2, we were also able to detect the SMIT2 protein (MW = 66 kDa) in Western blots of BBMV purified from SMIT2-transfected MDCK cells as well as in immunofluorescence studies, thus confirming expression at the apical domain in these cells. SMIT2 activity was also shown to be stimulated five-fold when submitted to 24 hours hypertonic treatment (+200 mosm). The SMIT2-MDCK cell line thus appears to be a promising model for studying SMIT2 biochemistry and regulation.