Bilirubin conjugates in the serum of newborn human infants were investigated using the alkaline methanolysis-high-performance liquid chromatography method, a specific and sensitive method for measurement of unconjugated bilirubin and bilirubin mono- and diester conjugates. Serum samples were analyzed from 13 premature infants, 11 full term newborns, 22 healthy adults, seven pregnant women at term and their corresponding infants cord blood at delivery, 46 cord blood specimens obtained at unselected deliveries, three cord bloods from infants with maternal-fetal blood group incompatibility, and two cord bloods from infants with intrauterine hypoxia. Bilirubin conjugates were not detectable in the healthy adults, maternal blood, or in the cord blood specimens except from infants with blood group incompatibility or intrauterine hypoxia. The two isomeric monoconjugates of bilirubin appeared in serum during the first 24 to 48 postnatal h in both premature and full term infants, followed by the diconjugate on the 3rd day. Conjugated esters accounted for 2 to 5% of the total bilirubin, with the diconjugate constituting 21% of total conjugated pigment (day 3). In all instances, the unconjugated serum bilirubin concentration had increased to at least 2 mg/dl in the course of physiologic neonatal hyperbilirubinemia before bilirubin conjugates became detectable. Both premature and full term human infants displayed the identical pattern of bilirubin conjugation in serum.