Previous work in this laboratory has suggested that the nonlinear disposition of valproic acid (VPA) in the rat may be due to nonlinear distribution of VPA into the liver. The present study was undertaken to elucidate further the hepatobiliary disposition of VPA. VPA (0.1-2 mmol/L) was incubated with isolated rat hepatocytes in vitro. Uptake of [(3)H]-VPA was linear from 10 to 50 seconds, with minimal (<7 percent) biotransformation. The initial velocity of VPA uptake varied in proportion with the extracellular concentration and was temperature independent, suggesting that VPA traverses the hepatocyte membrane predominantly by passive diffusion. In separate studies, the hepatobiliary disposition of VPA (20mg) was examined in the isolated perfused rat liver (IPL). A pharmacokinetic model was developed to describe the influence of phenobarbital on the hepatobiliary disposition of VPA and valproate glucuronide (V-G) in the IPL; all processes governing VPA and V-G disposition appeared to be linear. Acute administration of phenobarbital to the liver (1.12 mg) decreased the rate constant for canalicular egress of V-G (0.0489 +/- 0.0266 vs. 0.164 +/- 0.075 min(-1)). In vivo pretreatment with phenobarbital (75 mg/kg/d x 5 d) before liver isolation decreased the biliary excretion of both VPA (1.06E-04 +/- 0.27E-04 vs. 2.76E-04 +/- 0.45E-04 min(-1)) and V-G (5.63E- 03 +/- 1.98E-03 vs. 1.74E-02 +/- 0.5E-02 min(-1)), and increased the apparent volume of distribution of VPA (84.6 +/- 2.2 vs. 72.3 +/- 2.1 mL). In vivo phenobarbital pretreatment a changed V-G excretion from a formation to an elimination rate-limited process. These results are consistent with phenobarbital-associated impairment of canalicular egress of some organic anions. This work further supports the utility of pharmacokinetic modeling in: (1) determining the rate-limiting steps in hepatobiliary drug disposition and (2) identifying sites of drug interactions within the hepatobiliary system that may not be evident based on conventional mass-balance analysis.