Hepatic bilirubin conjugation and excretion were investigated during pregnancy and lactation in the rat. Bilirubin uridine diphosphate-glucuronyltransferase activity was decreased by 30% in pregnant rats, both when expressed per milligram of protein or as specific activity and per unit of liver weight. Liver size increased during pregnancy, and, as a consequence, total hepatic glucuronyltransferase activity was unchanged. The biliary bilirubin output was normal in pregnant rats, and when loaded with bilirubin, the maximal output in bile for the whole liver was also normal. In lactating rats, specific glucuronyltransferase activity returned to control values, but the activity per unit of liver weight was still lower, due to the decreased hepatic protein concentration. The liver remained enlarged during lactation, and total hepatic glucuronyltransferase activity was increased, together with the maximal output of bilirubin in bile. Two weeks after delivery, hepatic bilirubin conjugation and excretion in nonlactating mothers were comparable to those of virgin females. Parallel modifications of bilirubin glucuronyltransferase assayed in vitro and of maximal biliary output of the pigment in vivo were observed in all animals studied. The output of bilirubin diconjugates in bile was decreased during pregnancy but no changes of the proportion of the mono-to diconjugates in bile were observed 2 weeks after delivery both in lactating and in nonlactating rats. The modifications observed during pregnancy could not be reproduced by treatment with beta-estradiol and progesterone. This suggests that different hormones or modifications of steroid metabolism are probably involved in the alterations of hepatic bilirubin metabolism in pregnant and lactating rats.