Despite decades of preclinical investigation, there remains limited understanding of the etiology and biological underpinnings of anxiety disorders. Sensitivity to potential threat is characteristic of anxiety-like behavior in humans and rodents, but traditional rodent behavioral tasks aimed to assess threat responsiveness lack translational value, especially with regards to emotionally valenced stimuli. Therefore, development of novel preclinical approaches to serve as analogues to patient assessments is needed. In humans, the fearful face task is widely used to test responsiveness to socially communicated threat signals. In rats, ultrasonic vocalizations (USVs) are analogous social cues associated with positive or negative affective states that can elicit behavioral changes in the receiver. It is therefore likely that when rats hear aversive alarm call USVs (22kHz) they evoke translatable changes in brain activity comparable to the fearful face task. We used functional magnetic resonance imaging in male and female rats to assess changes in BOLD activity induced by exposure to aversive 22kHz alarm calls emitted in response to threatening stimuli, prosocial (55kHz) USVs emitted in response to appetitive stimuli, or a computer-generated 22kHz tone. Results show patterns of regional activation that are specific to each USV stimulus. Notably, limbic regions clinically relevant to psychiatric disorders (e.g., amygdala, bed nucleus of the stria terminalis), are preferentially activated by either aversive 22kHz or appetitive 55kHz USVs. These results support the use of USV playback as a promising translational tool to investigate affective processing under conditions of distal threat in preclinical rat models.Significance StatementAnxiety in humans often manifests as maladaptive responding to socially communicated threats. However, translational tools to study responses to negatively-valenced social stimuli in rodents are lacking. The fearful face task or similar paradigms are used in humans to indicate ambiguous distal or indirect threat and provide valuable information about critical nodes of brain activity via functional magnetic resonance imaging (fMRI). Rats use well-characterized ultrasonic vocalizations (USVs) as indices of affective states with communicative value; therefore, we tested whether playback of affectively valenced USVs could be leveraged as socially communicated ambiguous threat during fMRI in awake rats. Results support the use of USV playback as a promising translational tool to investigate affective processing in response to social cues.