[3H]Phenytoin binding to rat cortical membrane was significantly enhanced in the presence of diazepam. This binding is saturable, reversible and displacable by unlabelled phenytoin. Analyses of the binding data either by the Scatchard plot or by the displacement curve revealed a high and a low affinity sites with Kd values of 32 +/- 5 nM and 8.5 +/- 1.1 microM, respectively. Similar enhancement of [3H]phenytoin binding was observed when diazepam was replaced by Ro 5-4864 (4'-chlorodiazepam) which is selective for the 'peripheral' type benzodiazepine binding sites. In contrast, neither the 'central' type receptor selective agonist clonazepam nor the antagonist Ro 15-1788 enhanced [3H]phenytoin binding. Therefore, it seems that these phenytoin binding sites in rat cerebral cortex are associated with a benzodiazepine site similar to the 'peripheral' type binding site for its selective affinity for Ro 5-4864. However, judging from the micromolar concentrations required for the enhancement of [3H]phenytoin binding, they appear unlikely to be the same 'peripheral' type binding sites as measured by [3H]Ro 5-4864 binding (Kd approx. 1 nM). The micromolar affinity benzodiazepine recognition sites are a possibility, if they indeed exist.