The development of glial-enriched cultures prepared from 15-day-old embryonic chick cerebral hemispheres was examined after exposure to ethanol. Between culture days 6 and 10, ethanol was added to the medium at 4 concentrations: 0.1%, 0.5%, 1.0% and 2.0% (w/v). During the drug exposure interval, morphologic maturation of the cultures was surveyed with phase microscopy; cell proliferation was assessed by cell counts and DNA content. Exposure to ethanol concentrations of 0.1% and 0.5% resulted in only minimal changes in the parameters investigated, whereas biosynthetic and enzymatic activities were altered at a threshold dose of 1.0% ethanol. A small number of cells resembling reactive astrocytes were encountered in cultures exposed to 1.0% and 2.0% ethanol. In these cultures the number of cells and DNA content were lower than in control cultures. Additionally, [14C]leucine incorporation into protein and glutamine synthetase activity were markedly diminished in cultures treated with 1.0% and 2.0% ethanol. The ethanol-related reduction in glutamine synthetase activity was not accompanied by an equivalent decrease in protein content, suggesting that glutamine synthetase or cells rich in this enzyme are selectively vulnerable to ethanol. Impairment in glutamate/glutamine metabolism may be a potential consequence of ethanol-induced changes in glutamine synthetase activity.