myo-Inositol is a growth factor for mammalian cells as well as for the pathogenic protozoa Trypanosoma cruzi. Most of the cell surface molecules in this organism rely on myo-inositol as the biosynthetic precursor for phosphoinositides and glycosylated phosphatidylinositols. The aim of this work was to investigate the process of myo-inositol translocation across the parasite cell membrane. myo-Inositol uptake was concentration-dependent in the concentration range 0.1±10 mm with maximal transport obtained at 8 mm. Using sodium-free buffers, where Na 1 was replaced by choline or K 1 , myo-inositol uptake was inhibited by 50%. Furosemide, an inhibitor of the ouabain-insensitive Na 1 -ATPase, inhibited the Na 1 -dependent and Na 1 -independent myo-inositol uptake by 68 and 33%, respectively. In contrast, ouabain, an (Na 11 /K 1 ) ATPase inhibitor, did not affect transport. Part of the myo-inositol uptake is mediated by active transport as it was inhibited when energy metabolism inhibitors such as carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (34%), 2,4-dinitrophenol (50%), KCN (71%) and NaN 3 (69%) were added to the medium, or the temperature of the medium was lowered to 4 8C. The addition of glucose (5±50 mm) or mannose (10 mm) did not change the myo-inositol uptake, whereas the addition of 10 mm nonlabeled myo-inositol totally inhibited this transport, indicating that the transporter is specific for myo-inositol. Phloretin (0.3 mm) and phoridzin (5 mm), but not cytochalasin B, were efficient inhibitors of myo-inositol uptake. A portion of the accumulated myo-inositol is converted to inositol phosphates and phosphoinositides. These data show that myo-inositol transport in T. cruzi epimastigotes is mediated by at least two specific transporters Ð one Na 1 -dependent and the other Na 1 -independent.