OBJECTIVES: delta-Bilirubin (Bdelta) forms when bilirubin conjugates covalently bind to albumin by way of nonenzymatic transesterification in patients with cholestasis. Infants with cholestasis with biliary atresia form Bdelta. The aim of the present study was to investigate the factors determining serum Bdelta concentrations in infants with biliary atresia. METHODS: Study patients were infants enrolled in a prospective study (PROBE: Clinicaltrials.gov NCT00061828) of biliary atresia. We acquired data of concurrently measured serum bilirubin analytes (total bilirubin [TB], conjugated bilirubin [Bc], and unconjugated bilirubin) and applied graphical methods and linear mixed effects model to study factors contributing to Bdelta variability. RESULTS: Bdelta level increased with increasing levels of Bc and TB. In addition, the length of time cholestasis persisted partially determined the level of Bdelta. An increase of 1 mg/dL in Bc is related to approximately 0.36 mg/dL increase in Bdelta (P < 0.0001); every 100 days of cholestasis is associated with an approximately 1.0 mg/dL increase in Bdelta (P < 0.0001) given the same level of Bc. Serum albumin levels are not significantly related to Bdelta (P = 0.89). CONCLUSIONS: Bdelta levels in infants with biliary atresia increase with increasing levels of Bc and longer duration of cholestasis. Understanding the relation among Bdelta, Bc, TB, and direct-reacting bilirubin levels can help in interpretation of the clinical extent of cholestasis in infants and children with biliary atresia, assisting in the diagnosis and management of these infants.