Ultrasonic vocalizations (USVs) are essential communicative sounds used between rodent pups and their mother. Rat pups emit USVs in stressful situations, such as when they are cold or seperated from the nest. We verified the ontogenetic changes in USVs emitted by infant rats isolated from their mother during the pre-weaning period. The number of calls, and the median frequency and first peak of frequency of the calls were measured at 1, 3, 5, 7, 10, 12, and 14 days postnatal age in Wistar-Imamichi rats. Pups were placed in a cold glass beaker and USVs were recorded for 5 min. The number of calls increased to a peak on day 5 and then gradually decreased. The median frequency of calls decreased slowly during the first 12 days, and then increased slightly on day 14. Similarly, the first peak frequency of calls was the highest on day 1, and then decreased gradually by day 12. A small increase was observed on day 14. These changes in frequency were correlated with the physical development of the pups, whose body weights increased significantly with age except during postnatal days 7-10.