The unbound “free” bilirubin concentration (Bf), not the total bilirubin concentration, is the critical determinant of cellular uptake and toxicity of bilirubin. We compared Bfmeasured by a modified peroxidase method with published data obtained with ultrafiltration and examined conditions that affect the affinity (KF) of human (HSA) and bovine (BSA) serum albumin for bilirubin. The peroxidase and ultrafiltration methods yielded similar KFvalues that decreased with increasing HSA concentration and the presence of 50 mM chloride. When related to ionic strength, inhibition of BSA-bilirubin binding by chloride, bromide, and sulfate were similar, whereas phosphate buffer had a smaller effect. KFwas lower at 37°C than at 25°C for HSA but not for BSA. KFfor BSA was similar at pH 7.4 and 8.0. BSA and FCS had similar binding properties. The close agreement of Bfand KFvalues determined by the peroxidase method with published results obtained by ultrafiltration validates both methods and supports the use of the peroxidase method as a practical technique for measuring Bfunder steady state conditions in minimally diluted serum or culture medium.