The effect of valproate on the pharmacokinetics of phenytoin after a single i.v. injection is described in this study. Valproate is shown to significantly increase the total clearance, i.e., 23.8 to 30.4 ml . min-1 . kg-1 and volume of distribution, i.e., 0.92 to 1.25 liter . kg-1. No significant change was shown for the half-life of phenytoin. The pharmacokinetic parameters calculated for unbound phenytoin showed no significant differences between those rats infused with phenytoin or phenytoin-valproate. Significantly higher concentrations of 5-(p-Hydroxyphenyl)-5-phenylhydantoin (HPPH) were observed in those rats given an i.v. injection of phenytoin-valproate. In vitro studies with rat liver microsomes demonstrated that valproate inhibits the metabolism of HPPH to its glucuronide. This was further exemplified when significant differences were observed in the clearance and volume of distribution of HPPH in rats given an i.v. injection of HPPH or HPPH plus valproate. The results of this study demonstrate that in the rat valproate displaces phenytoin from serum proteins and that this interaction accounts for the increased volume of distribution and clearance of total phenytoin when valproate is coadministered. However, this displacement had no effect on the pharmacokinetics of free (unbound) phenytoin. Valproate is shown to be an inhibitor of the glucuronidation of HPPH which may explain the significantly increased concentrations of HPPH observed in the rats administered phenytoin-valproate.