The present study investigated how frequency distribution of maternal separation-induced ultrasonic vocalization was altered by environmental stimuli and pharmacological agents. Sprague-Dawley rat pups at 10 days were used to measure numbers and frequencies of the ultrasonic vocalizations under various ambient temperatures and with pharmacological manipulations of the corticotropin-releasing factor (CRF) and GABAergic systems. The ultrasonic vocalization consisted of two distinct peaks in the frequency range of 30 kHz to 50 kHz. The area under the curve (AUC) in the high-frequency range and the number of the ultrasonic vocalizations increased when ambient temperature was lowered. Systemic administration of a selective CRF1 receptor antagonist, NBI27914, and a typical anxiolytic, diazepam, decreased the AUC in the high-frequency range and the number of the ultrasonic vocalizations in a dose-dependent manner at an ambient temperature of 24 degrees C. The AUC in the low-frequency range did not change with an alteration in ambient temperature or treatment with NBI27914 and diazepam except a high dose (1 mg/kg) of diazepam. These results demonstrated that emotional levels of isolated pups reflected not only the number but also the frequency distribution of maternal separation-induced ultrasonic vocalizations. High-frequency components, but not low-frequency components, of the ultrasonic vocalization were sensitive to changes in negative affective state of isolated pups.