D-chiro-Inositol is an epimer of myo-inositol that is found in certain mammalian glycosylphosphatidylinositol protein anchors and inositol phosphoglycans possessing insulin-like bioactivity. In order to generate a probe for metabolic studies, D-chiro-[3-3H]inositol was synthesized by selective reduction of D-chiro-3-inosose at pH 6.5 with sodium borotritide. D-chiro-[3-3H]Inositol was taken up by HepG2 human liver cells through a saturable and stereospecific pathway in which D-chiro-inositol and myo-inositol competed equally but L-chiro-inositol was not recognized. Dd-Glucose, but not L-glucose, competed for D-chiro-[3-3H]inositol uptake over glucose concentrations of 4-28 mM. Maximum transport capacity was 717 pmol/mg cell protein/3 h with a Km value of 348 microM. Uptake was reduced by 76% when sodium was eliminated from the medium and by 94% when the experiment was performed at 0 degrees C. The new myo/D-chiro-inositol transporter is distinct from the sodium-myo-inositol co-transporter found in many tissues and accounts for all of the saturable D-chiro-inositol uptake and for a portion of the saturable low affinity myo-inositol uptake in HepG2 cells. It may allow D-chiro-inositol to be used by cells in the presence of a relatively large amount of competing myo-inositol.