Gall bladder storage of hepatic bile prevents complete recovery of biliary excretion of drugs to be obtained under physiological conditions in man. The aim of this study was to develop and validate a method for simultaneous measurement of gall bladder storage of a cholephilic drug, and of its duodenal excretion and t1/2 in bile. Duodenal perfusion using polyethylene glycol as intestinal recovery marker for measurement of drug duodenal excretion, with an iv bolus of 99mTc HIDA for measurement of drug mass within the gall bladder was used. Gall bladder volume was measured by ultrasonography. T1/2 in bile was measured by relating drug duodenal excretion to that of bile acid used as an endogenous bile marker. The use of bile acid as biliary marker was validated in two subjects receiving simultaneous iv infusion of indocyanine green. Seven healthy subjects were studied using a beta-lattam antibiotic, Cefotetan 1 g iv, as test drug. Median values during the study period (seven hours) were 51.1 mg for Cefotetan duodenal excretion, 45.2 mg for gall bladder mass and 2.8 mg/ml for concentration within the gall bladder. T1/2 of the drug in bile was 100 minutes. This technique enables measurement of mass and concentration of drugs within the gall bladder to be carried out, in addition to measurements of t1/2 of drugs in bile. These measurements may have specific application for assessment of potential efficacy of antibiotics in biliary tract infections, as well as general application for assessment of biliary excretory kinetics of drugs.