The effects of ethanol on hexose uptake by glial cells was investigated using primary cultures prepared from term rat fetuses. Specific 3H 2-deoxy-D-glucose (2DG) uptake was significantly reduced by a 4-hr exposure to ethanol at concentrations of 25, 50, and 100 mM, but not 200 or 300 mM. The inhibitory effect of 50 mM ethanol increased with the duration of exposure, with 2DG uptake inhibited by 36% after 18 hr. Astrocytes cultured from the brains of term fetuses of rats fed ethanol during pregnancy showed essentially the same 2DG uptake response to in vitro ethanol treatment. Kinetics of 2DG uptake showed a significant decrease of Vmax in the presence of ethanol. No interaction was found between ethanol and insulin, which stimulated 2DG uptake and protein content of the cultures. The data suggest that ethanol can modulate hexose uptake by astrocytes cultured from fetal rat brain. However, insulin actions on glial cells were not affected by ethanol.