To elucidate the role of opioid peptides in control of the anxiety-like behavior and anxiety-reducing actions of benzodiazepines, a recombinant, replication-defective herpes virus (SHPE) carrying human preproenkephalin cDNA was delivered to rat amygdala. Viral infection resulted in a strong, localized transgene expression after 2-4 days which diminished after one week. Anxiety-like behavior and the anxiolytic effect of diazepam were assessed three days after gene delivery using the elevated plus maze test. While SHPE infection alone did not reduce anxiety-like behavior, rats infected with SHPE exhibited a greater response to the anxiolytic effect of diazepam when compared to rats infected with a control virus (SHZ.1) containing the lacZ gene. The enhancement of diazepam action by SHPE was naloxone reversible, region-specific, and correlated with the time course of preproenkephalin expression. The findings implicate amygdalar opioid peptides in regulating the anxiolytic effects of benzodiazepines. This study also demonstrates the usefulness of recombinant herpes virus in evaluating the role of single gene products within specific brain sites in pharmacological responses and complex behaviors.