Sugar uptake was measured in dispersed cells prepared from radiation-induced insulinomas transplantable in NEDH rats and in three clonal β-cell lines maintained in continuous culture (RIN m5F, RIN 1046, HIT). Uptake of D-glucose and 3-O-methyl-D-glucose by insulinoma cells was rapid so that the intracellular concentration of D-hexoses approximated the concentration in the incubation medium by 15–30 s. L-Glucose was taken up only slowly. 3-O-methyl-D-glucose uptake by RIN m5F, RIN 1046, and HIT cells was slow; with 1 mM 3-O-methylglucose in the medium, equilibrium was attained at 20 min, but with 10 mM 3-O-methylglucose, equilibrium was not attained even at 20 min. In HIT cells incubated with D-glucose for 30 min, the intracellular concentration of glucose was less than the medium glucose concentration, indicating glucose transport is a nonequilibrium reaction in this cell line. These data indicate that radiation-induced insulinoma cells retain the capacity of normal β-cells to transport sugar at high rates. RIN m5F, RIN 1046, and HIT cells transport sugar slowly, however, and thus differ from normal β-cells. In RIN m5F, RIN 1046, and HIT cells, unlike in normal β-cells, glucose transport may be the site regulating glucose metabolism.