Utilizing psychogenetically selected Roman high- and low-avoidance rats (RHA/Verh and RLA/Verh), the present experiments investigated the effects of prenatally administered vehicle and diazepam (1 and 3 mg/kg per day, SC) on the behavior and neurochemistry of adult, male offspring. Active, two-way avoidance behavior was analyzed in 96 rats, at 6 months of age, and swimming navigation in 68 others, at 11 months. Three weeks after testing, selected brain areas from the latter animals were immunoassayed for benzodiazepine (BZD)-like molecules. The 3 mg/kg dose of diazepam both decreased freezing behavior in the shuttle box and reduced the hippocampal content of BZD-like molecules in the RLA/Verh male rats. Swimming navigation (spatial learning), at which the RLA/Verh rats were more adept, was not specifically affected by prenatal diazepam in either rat line. The possibility exists that an increased hippocampal release of BZD-like substances may be necessary to alter shuttle box behavior in RLA/Verh rats.