Agonists acting at benzodiazepine, gamma-aminobutyric acidA, barbiturate and neurosteroid recognition sites were studied for their attenuation of separation-induced ultrasonic vocalizations (USV) in rat pups. The behavioral effects of the neuroactive steroid 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) were assessed when the drug was administered alone and in combination with agonists and antagonists acting at the gamma-aminobutyric acidA receptor complex. At 7 days postpartum, male and female Long-Evans rat pups were separated from the dam and littermates, and placed on a 20 degrees C surface for 2 min. Allopregnanolone (1-30 mg/kg s.c.), alprazolam (0.03-1 mg/kg s.c.), diazepam (0.1-3 mg/kg s.c.), muscimol (0.03-0.3 mg/kg s.c.) and pentobarbital (1-30 mg/kg s.c.) dose-dependently decreased USV. Pretreatment with flumazenil (0.1 mg/kg s.c.) antagonized alprazolam's and diazepam's USV-suppressive effects; bicuculline (2 mg/kg s.c.) reversed muscimol's USV-suppressive effects. Allopregnanolone (3 mg/kg s.c.) produced a 4- to 7-fold leftward shift in alprazolam's and diazepam's USV-suppressive effects, and also produced a modest leftward shift in pentobarbital's USV dose-effect function. Neither flumazenil, bicuculline, nor picrotoxin (1 mg/kg s.c.) altered allopregnanolone's USV-suppressive effects. These results suggest that the USV-suppressive effects of the neurosteroid allopregnanolone are mediated at the gamma-aminobutyric acidA receptor complex, and are independent from a direct action on the benzodiazepine or gamma-aminobutyric acidA recognition sites on this complex.