The biliary excretion of cefazolin was compared with that of cephalothin and cephaloridine in rats and man. In rats, the biliary levels were dose-related with cefazolin and cephalothin but not with cephaloridine. Biliary levels were higher than serum levels after injection of 10–80 mg/kg of cefazolin and cephalothin, whereas serum levels of cephaloridine after injection were higher than biliary levels. The highest biliary levels of cefazolin were obtained by intravenous injection, followed by intramuscular injection and drip infusion. In man, a crossover study was made to compare biliary levels of cefazolin with those of cephaloridine and cephalothin. After a single 1-gram intravenous injection, the peak levels of cefazolin ranged from 0.85 to 21 μ g/ml and those of cephaloridine varied from 0.55 to 3.9 μ g/ml. After a 3-gram intravenous injection, the peak biliary levels of cefazolin ranged from 35.5 to 270 μ g/ml and those of cephalothin from 0.3 to 64 μ g/ml. The chemotherapeutic biliary levels of cefazolin able to inhibit susceptible organisms were obtained by 3-gram intravenous injections.