Disruption of the primate mother-infant attachment bond is a naturally occurring stressor that results in marked behavioral, physiological, and endocrine activation. We studied the effect that altering benzodiazepine systems has on the behavioral and endocrine response of infant rhesus monkeys (1-27 weeks of age) to brief separation from their mothers. In the first experiment, the benzodiazepine agonist diazepam (0.1 and 1.0 mg/kg) significantly increased locomotion and social behavior and decreased inactivity and distress vocalizations in infant monkeys undergoing separation. In the second experiment, the benzodiazepine antagonist Ro 15-1788 (5 and 10 mg/kg) had no significant effects on the infants' separation response. In the third experiment, administration of diazepam 1.0 mg/kg was followed by administration of Ro 15-1788 10 mg/kg in infants undergoing separation. Ro 15-1788 blocked the decreases both in inactivity and in plasma ACTH and cortisol concentrations caused by diazepam, suggesting that these effects are mediated through benzodiazepine receptors. These data support the hypothesis that in primates, endogenous benzodiazepine systems modulate the behavioral and endocrine response to the naturally occurring stress of separation.