Biliary obstruction has been recognized to inhibit excretion of antibiotics into bile. In the present study, using cefpirome sulfate (CPR), we sought to determine the effect of biliary pressure on antibiotic transfer into bile in patients undergoing endoscopic retrograde cholangiopancreatography (ERCP). Thirty‐six patients with a variety of biliopancreatic diseases (free of renal disease or hypoproteinemia) received a single intravenous dose of CPR (1 g) prior to ERCP. Under fluoroscopy a diagnostic catheter with a metal ball tip was advanced into the middle portion of the extrahepatic bile duct or, in cases of common bile duct obstruction, above the obstruction. Biliary pressure was measured via the same catheter using duodenal pressure as a reference. Subsequently, bile was aspirated, and blood was withdrawn simultaneously. The mean interval between CPR administration and the bile and blood samplings was 67±12 minutes. The bile CPR concentration and the bile/serum ratio of CPR concentrations showed a significant inverse correlation with biliary pressure, but the serum CPR concentration did not. The bile CPR concentration and the bile/serum ratio of CPR concentrations differed significantly between the group with normal biliary pressures, below 10 mmHg, and that with biliary pressures exceeding 10 mmHg. The serum CPR concentrations of the two groups were similar. These results suggest that biliary pressure plays an important role in determining antibiotic transfer into bile.