The 24h urinary bile acid excretion was prospectively studied during the neonatal period in healthy, fully breastfed, premature and full-term infants. The urinary bile acids were identified by gas-liquid chromatography (GLC)-mass spectrometry and quantified by GLC. The excretion of bile acids in urine increased after birth, reaching maximum levels by the 3-4th day. Taurine conjugates predominated and the excretion of bile acid sulphates was remarkably low. Cholic acid and atypical bile acids were the main bile acids in urine during the first week. Tetrahydroxylated bile acids carrying hydroxyl groups at C-1, C-2 and C-6 were common, and also other 1- and 6-hydroxylated bile acids, including hyocholic and hyodeoxycholic acids. Three tentatively identified 4-hydroxylated bile acids, including one ketonic bile acid, were also found. Ketonic bile acids constituted an average of 16% of total urinary bile acids during the first week. Unsaturated bile acids were scantily found only during the first days. The excretion of atypical bile acids decreased to 1 month of age, parallel with the total bile acid excretion. The data support earlier hypothesis of a physiological cholestasis in the newborn. Atypical hydroxylated and ketonic bile acids, as well as cholic acid, constituted the major part of the urinary bile acids. The persistent atypical pattern of bile acids in urine during the first month of life indicates a longer period of immaturity of bile acid metabolism in healthy infants than previously described.