The ontogenetic changes in responsiveness to benzodiazepine receptor ligands on ultrasonic vocalizations in rat pups from the age of day 3 to day 12 were evaluated. Rat pups, while separated from their dam and littermates and placed in a cold environment, emit ultrasonic vocalizations. These ultrasonic calls became attenuated dose-dependently in number and power after administration of the anxiolytic diazepam (0.25-1.0 mg/kg, s.c.), but the inhibitory effect of diazepam at the highest dose was less on day 6 and day 9. Moreover, type 1 benzodiazepine receptor ligands, Ro16-6028 and Ro23-0364 (0.5-2.0 mg/kg, s.c.), also dose-dependently attenuated the ultrasonic vocalizations 30-60 min after injection. The inhibitory effects of these drugs became more pronounced with the increasing age of the pup, and they were equivalent on day 12 to those in adult rats. These results suggest that different ontogenetic changes in development of two subtypes of central benzodiazepine receptors of pups might be related in the psychopharmacological mediation of the ultrasonic vocalization.