Many xenobiotics cause hepatobiliary toxicity and cholestasis in the rat. Initial assessment of hepatobiliary damage in rats can be accomplished by measuring serum concentrations of bile acids and bilirubin, serum activities of liver-associated enzymes such as 5'-nucleotidase, alkaline phosphatase, gamma-glutamyltranspeptidase, and plasma clearances of dyes [e.g., bromosulfophthalein (BSP)] excreted primarily through the bile. More detailed evaluation of hepatobiliary disturbances involves cannulation of the bile duct of anesthetized rats and subsequent measurement of rates of bile flow, bile acid excretion, and bile composition. Canalicular bile flow can be estimated from clearances of nonmetabolized sugars (i.e., erythritol) which enter bile via paracellular transport. Tight junction permeability also can be assessed by either biliary excretion of such a marker as horseradish peroxidase or sucrose following portal vein infusion or via retrograde biliary infusion. Subsequent morphologic evaluation of the liver provides information on damage to cells which may contribute to hepatobiliary dysfunction (i.e., bile duct obstruction). Isolated perfused livers offer the ability to measure all of the above mentioned parameters as well as to make a more accurate determination of the effects of xenobiotics on bile acid-dependent and -independent bile flow. A good example of the advantage of combining techniques as well as following complete time courses of changes in hepatobiliary function is provided by using studies of alpha-naphthylisothiocyanate-induced hepatotoxicity.