During playful interactions, rats emit increased levels of 50-kHz vocalizations. It is possible that these vocalizations are used as play signals that promote and maintain playful contact. The study investigated this possibility. It was predicted that if these vocalizations are used as play signals, they should be more prevalent (1) before an attack, (2) in attacks leading to wrestling, and (3) in males compared to females, as males play more roughly. Moreover, given that there are at least 15 different subtypes of 50-kHz calls, it is possible that different calls are used in different contexts. Therefore, our prediction (4) was that different subtypes would be used for initiating and terminating playful contact. Pairs of same-sex juveniles were tested so that video recordings of their play and audio recordings of their vocalizations were synchronized. 50-kHz vocalizations occur more often before an attack and in male pairs. Specific calls were associated with specific types of behaviors and these associations differed between male and female rats. However, calls were not more frequent in attacks leading to wrestling than in attacks leading to withdrawal. The data provide qualified support for the hypothesis that 50-kHz vocalizations function as play signals. © 2014 Elsevier B.V.