Rat pups, while separated from their littermates and placed in a novel environment, emit ultrasonic isolation calls. These ultrasonic calls decrease in number, power, and frequency following administration of the anxiolytic, diazepam (0.5 mg/kg). Pentylenetetrazol (20 mg/kg), which has been reported to be clinically anxiogenic, increases the number and the power of these calls. These changes following diazepam and pentylenetetrazol administration are dose dependent and do not appear to be secondary to nonspecific effects of these drugs on arousal or thermoregulation. The benzodiazepine receptor antagonist RO 15-1788, which has generally been reported to lack intrinsic activity at low doses, also decreases the number of rat pup isolation calls. These findings suggest that the benzodiazepine-GABA receptor-chloride channel complex may play a role in the physiologic mediation of the rat pup isolation call.