Pharmacokinetic analyses of three kinds of benzodiazepines--midazolam (MDZ), triazolam (TRZ) and alprezolam (APZ)--were performed in rats with cannulated portal and jugular veins. Each drug was administered to the double-cannulated rats, and pharmacokinetic data for the parent drugs and their 1'- and 4-hydroxylated metabolites were compared with those obtained in non-cannulated mice. In bioavailability, the drugs ranked APZ >> TRZ = MDZ in rats, and APZ > TRZ >> MDZ in mice, with the values for MDZ remarkably different between rats and mice (19% in rats versus 2.3% in mice). In contrast, hepatic availability (Fh) was similar (APZ > TRZ > MDZ) in both species. Highly significant relationships were found between the ratio of the area under the plasma concentration-time curve (AUC) for the parent drugs in portal blood (AUC(por)) to that in systemic blood (AUC(sys)) and Fh in rats and mice. The double-cannulated rat is useful for estimating the hepatic availability of drug candidates by determining the AUC values for the parent drugs in portal and systemic blood samples.