The pathways of glucose metabolism in fetal rat brain have been studied by determination of the enzymatic activities in cell-free extracts and by isotopic tracer techniques. Cell-free extracts of both fetal and adult rat brain contained enzymes for glucose metabolism through the Embden-Meyerhof pathway and the pentose phosphate cycle. Enzymes for glycolysis were several times more active than the enzymes of the hexose monophosphate pathway in both tissues, but in fetal rat brain the pentose phosphate shunt enzymes were significantly more active than in adult brain. Tracer studies were carried out with glucose labeled in various positions to estimate the yield of 14CO2, the corrected specific activity of lactate formed from [1-14C]- and [6-14C]glucose, the randomization of the labeled carbon of [2-14C]glucose in the other carbons of lactate and the effect of pyruvate on the yield of 14CO2 from [1-14C]- and [6-14C]glucose. Results indicate that in fetal rat brain glucose is mainly metabolized through the Embden-Meyerhof pathway, but the pentose phosphate cycle participates to a greater extent in fetal brain than in adult brain. © 1967.