Myo-inositol transport and metabolism were studied in cultured human skin fibroblasts exposed to potentially toxic levels of glucose or galactose. Although variable among 11 different cell lines, the myo-inositol level in confluent cells, ranging from 10–50 nmol/mg protein, was constant with passage. A high-affinity transport system for myo-inositol had an apparent K, of 55 μM and Vmax of 16 pmol/min/mg protein. No obvious relationship existed between cellular levels and transport capacity. Dependency on sodium was complex. When medium sodium was lowered to 23 mM, myo-inositol uptake ceased after about 1 h. However, the initial rate of myo-inositol uptake only showed a sodium dependence at low myo-inositol concentrations. Both phloretin and phloridzin inhibited myo-inositol uptake. Phloridzin had a K1 of 60 μM, and phloretin was either a noncompetitive or uncompetitive inhibitor. Glucose and galactose were only weak competitive inhibitors, with a K1 of 30 mM and 65 mM, respectively. After 24 h of incubation with myo-[2-3H]inositol, only 10% of the total cell label was incorporated into phospholipid. Compared with control media with 5 mM glucose, the incubation of confluent cells in media with 20 mM glucose had little effect on intracellular glucose and sorbitol, whereas cells incubated in control media supplemented with 5 mM galactose showed a large increase in galactose and polyol levels. In media with more than 200 μM of myo-inositol, neither treatment had an effect on wyo-inositol levels after 24 h. The uptake and incorporation of 11 μM myo-[2-3H]inositol and incorporation into phospholipid were studied after cells had been previously exposed for 24 to 48 h to media supplemented with 15 mM glucose or galactose. Compared with controls, fibroblasts with a 24-h exposure to 20 mM glucose showed a 10% decrease in myo-inositol uptake. When the exposure was extended to 48 h, preconditioning with galactose as well as glucose elicited the same 10% reduction in uptake. Phosphoinositide labeling in fibroblasts exposed to 20 mM glucose was reduced in parallel. These cells offer a unique opportunity for the study of sugar toxicity in human tissue: they can be exposed to high levels of glucose without significant glucose or polyol accumulation or can be made to accumulate polyol by exposure to moderate levels of galactose. The expression of a hexose-induced reduction in myo-inositol transport required 24 to 48 h of exposure of the fibroblasts to elevated concentrations of glucose or galactose and may not be related to a competitive inhibitory effect of these sugars on transport.