Bumetanide binding to human serum albumin was studied using ultrafiltration. The first stoichiometric binding constant for bumetanide is 6.4 X 10(4) M-1. Bumetanide competes with bilirubin for human serum albumin binding, having a KDispl (displacement constant) of 6.2 X 10(3) M-1 measured by the peroxidase method. This displacement effect is also observed using pooled umbilical cord serum and pooled adult serum employing a dialysis rate method. Bumetanide competes to a lesser degree with diazepam binding to human serum albumin. No competition with diazepam occurs using umbilical cord or adult serum. Pharmacologic concentrations of bumetanide would not significantly affect bilirubin-albumin binding and should not increase the risk of bilirubin encephalopathy in newborn infants.