Knowledge of the reserve bilirubin-binding capacity is useful in management of hyperbilirubinemia in the newborn. We describe thin-layer gel chromatography that permits measurement of this variable, with a 250-mul sample of serum, within 50 min. Single samples from four different infants can be analyzed on the same plate. The capacity of 40 sera from jaundiced and of 19 sera from nonjaundiced infants was measured. Results by thin-layer Sephadex gel chromatography and column Sephadex chromatography correlated well. Dithiothreitol, added to the standard bilirubin solution, protected bilirubin from oxidation for at least six days, which obviated the time consuming preparation of standard solution before such determination.