Microsomal galactosyltransferase activity of fetal rat colon increased fourfold between 18 and 22 days of gestation and then more slowly during neonatal life reaching adult levels after 14 days. The Km for uridinediphosphate- (UDP) galactose, pH optimum, cation, and detergent requirements were identical in fetal and adult galactosyltransferase. Cytidine 5'-diphosphate-choline stimulated the adult but not fetal colonic galactosyltrasferase activity by inhibition of UDP-galactose pyrophosphatase. The increase in colonic galactosyltransferase in late fetal development is correlated with our previous observation that incorporation of [3H]galactose is markedly increased during differentiation of the fetal colon.