Filtered myo-inositol, an important renal intracellular organic osmolyte, is almost completely reabsorbed. To examine tubule sites and specificity and, thus possible mechanism of this reabsorption, we microinfused myo-[³H]inositol or D-[³H]glucose into early proximal (EP), late proximal (LP), or early distal tubule sections of superficial nephrons and into long loops of Henle (LLH) of juxtamedullary nephrons and papillary vasa recta in rats in vivo et situ and determined urinary fractional recovery of the ³H label compared with comicroinfused [¹⁴C]inulin. To determine the extent to which the proximal convoluted tubule (PCT) alone contributes to myo-inositol reabsorption, we also microperfused this tubule segment between EP and LP puncture sites. We examined specificity of reabsorptive carrier(s) by adding high concentrations of other polyols and monosaccharides to the infusate. The results show that >60% of the physiological glomerular load of myo-inositol can be reabsorbed in the PCT and >90% in the short loop of Henle (SLH) by a saturable, phloridzin-sensitive process. myo-Inositol can also be reabsorbed in the ascending limb of LLH and can move from papillary vasa recta blood into ipsilateral tubular structures. Essentially no reabsorption occurred in nephron segments beyond the SLH or in collecting ducts. Specificity studies indicate that reabsorption probably occurs via a luminal Na⁺-myo-inositol cotransporter.