The association between high-ambient glucose, the polyol pathway, and aldose reductase inhibition on in vitro myo-[3H]inositol uptake was examined in cultured bovine lens epithelial cells (BLECs). Myo-[3H]inositol accumulation in the presence of 5.5 mmol/l D-glucose was rapid and linear for 8 hr. When Na+ was replaced on an equal molar basis with N-methyl-D-glucamine chloride, myo-[3H]inositol uptake was reduced by more than 95%. The myo-inositol transport system appear to be distinct from glucose transport, based upon three criteria: (1) 2-deoxy-D-[3H]glucose uptake, unlike myo-[3H]inositol uptake, was largely sodium independent; (2) L-glucose was a competitive inhibitor of myo-[3H]inositol uptake but had no effect on 2-deoxy-D-[3H]glucose uptake; and (3) 2-deoxy-D-[3H]glucose uptake appeared independent of myo-inositol concentration. Sodium-dependent myo-[3H]inositol uptake was substantially inhibited after chronic (20 hr) exposure of cultured cells to 40 mmol/l glucose. Inhibition of aldose reductase activity partially prevented the inhibitory effect of glucose on myo-[3H]inositol accumulation. No significant difference in the rates of passive efflux of myo-[3H]inositol from preloaded high glucose-treated and control cultures was observed. Although the coadministration of sorbinil to the high-glucose medium partially protected against the attendant decrease in transport activity, the failure to normalize myo-[3H]inositol uptake suggested that glucose-sensitive and sorbitol-sensitive processes were involved in the uptake of myo-inositol.