Metabolism of galactose was examined in dissociated brain cells from neonatal mice after 10-13 days in culture. Consumption of galactose at levels up to 26 mM was much less than consumption of glucose at corresponding concentrations. Lactate was consumed from the media at all galactose levels, in contrast to experiments with glucose in which lactate was formed and released into the media. Generation of CO2 from 4 mM glucose was 9-fold greater than from an equimolar level of galactose. Relatively low concentrations of glucose could reduce uptake of galactose, whereas galactose at levels up to 11.6 mM failed to inhibit consumption of glucose or formation of lactate. In glucose-deficient states, galactose supplementation of the media led to a marked increase in sulfatide synthesis by oligodendrocytes in the culture with a maximum effect at 2.3 mM. Under these conditions, [1-14C]galactose was incorporated directly into the carbohydrate portion of sulfatide, although most of the label was found in phospholipids and in the nonlipid fraction of the cellular homogenate. These data suggest that galactose is poorly metabolized by brain cells, but does not exhibit toxic effects.