Rat pups produce ultrasonic vocalizations (USVs) on isolation from their dam. Ultrasonic vocalization is a sensitive tool for evaluating social behavior between pups and their dam. Prenatal ethanol-exposure leads to a reduction in USVs and have the potential of inducing difficulties in social behavior between pups and their dam. However, effects of prenatal ethanol-exposure on the acoustic characteristics of USVs remain unclear. In this study, we recorded USVs produced by rat pups that were prenatally exposed to ethanol and examined their acoustic characteristics. Ethanol was administered to 13 pregnant rats in three stages by gradually increasing concentrations between gestational days (GDs) 8-20. From GDs 14-20, ethanol-containing tap water at concentrations of 30% and 15% (v/v) was administered to the high- and low-ethanol groups, respectively. Tap water without ethanol was given to the control group. On postnatal days (PNDs) 4, 8, 12, and 16, individual newly-born pups were isolated from their dam and littermates and USVs produced by them were recorded for 5 min. The number of USVs in the high-ethanol group was greater than that in both low-ethanol and control groups on PND 12. The mean, minimum, and maximum fundamental frequencies of USVs were elevated in the high-ethanol group compared with that in both low-ethanol and control groups. Higher amplitudes of USVs were produced by male pups in the high-ethanol group than in those in both low-ethanol and control groups on PND 12. These results suggest that prenatal ethanol exposure changed emotionality and accordingly, the high-ethanol group produced more USVs as distress calls.