Abstract The steroid N-acetylglucosaminyl transferase of rabbit liver has been found also in rabbit kidney and intestine. It is absent from uterus, ovary, adrenal, spleen, and blood. In kidney the activity is higher than that of glucuronyl transferase, but this relationship is reversed in the other tissues. The transferase is located in the microsomes, requires UDP-N-acetylglucosamine, has a pH optimum of 8.0 in phosphate buffer, and a Km value of 6.8 x 10-5 m for 17α-estradiol-3-glucuronoside. It will also transfer N-acetylglucosamine to the 3-glucuronosides of 17-epiestriol and 16,17-epiestriol, but not to those of estriol or 16-epiestriol. Of a series of free steroids tested, including 17α-estradiol, 17-epiestriol, and 16,17-epiestriol, none served as substrate for the enzyme, although estrone, as well as diethylstilbestrol and some aliphatic alcohols, inhibited its activity. The results indicate a high specificity of the enzyme for the 17α-hydroxyl group on phenolic steroids which are already attached through position 3 to glucosiduronic acid.