The product of the in vitro reaction catalyzed by bilirubin UDPglucuronyl-transferase has been studied with an improved chromatographic procedure for the separation and measurement of the diazo pigments. The source of enzyme activity was total microsomes and the smooth and rough microsomal subfractions after dialysis against EDTA. Biliburin monoglucuronide was the only conjugate formed by smooth microsomes while total and rough microsomes formed bilirubin mono and diglucuronide. These findings are compatible with the presence in rough microsomes of a factor(s), not present in smooth microsomes, necessary for the formation of bilirubin diglucuronide. The activity of bilirubin UDPglucuronyltransferase in rough microsomes was higher than in the smooth fraction.