Primary bile acid concentrations were measured in serum of 332 newborns with neonatal hyperbilirubinemia (serum total bilirubin level > 200 μmol/l) and compared with those of 95 nonhyperbilirubinemic neonates (serum total bilirubin level < 200 μmol/l). The serum concentrations (μmol/l; mean ± SEM) for cholic acid (8.78 ± 0.44) and chenodeoxycholic acid (10.5 ± 0.68) were significantly higher (p < 0.001) in the hyperbilirubinemic group than in the controls (7.16 ± 0.48 and 6.67 ± 0.48, respectively). 80 (24%) of the hyperbilirubinemic newborns had true cholestasis (serum levels of cholic and/or chenodeoxycholic acid higher than mean +2 SD in the reference group). The ratio of cholic to chenodeoxycholic acid was significantly higher (p < 0.05) in the cholestatic group than in the hyperbilirubinemic newborns without cholestasis. There was no significant difference in the serum concentrations of alkaline phosphatase or lactate dehydrogenase between the cholestatic and noncholestatic groups. In the hyperbilirubinemic newborns, the primary bile acids were indiscriminately raised. Only 8 infants from the 332 newborns had jaundice at the age of 1 month. Of these 8 infants only 2 had neonatal cholestatic hyperbilirubinemia. It thus appears that measurement of serum primary bile acid concentrations has only limited diagnostic value in assessing the severity or prognosis of neonatal hyperbilirubinemia.