In rat pups, ultrasonic vocalizations were emitted in response to separation from the mothers, littermates, and nest. It has been suggested that these separation-induced ultrasonic vocalizations (SIV) in rat pups form one of the animal models of anxiety. The primary aim of the present study is to investigate the effect of the compounds acting on stress-related peptide receptors such as a vasopressin V1b receptor antagonist and a corticotropin-releasing factor CRF1 receptor antagonist in rat pup SIV. The secondary objective is to establish further confirmation of the predictive validity of SIV testing. Both the V1b receptor antagonist SSR149415 and the CRF1 receptor antagonist CP154,526 reduced SIV, suggesting antagonists for stress-related peptide receptors are effective in this model. Furthermore, as with selective serotonin reuptake inhibitors such as fluvoxamine and paroxetine, SIV was also reduced by the serotonin and noradrenaline reuptake inhibitor milnacipran and the metabotropic glutamate receptor 5 antagonist MPEP, while desipramine was without effect. Thus, the present experiment highlights the important role of the stress-related peptide systems as well as of the serotonergic systems in SIV. Moreover, the present data support the usefulness of SIV for evaluating the anxiolytic-like activity of mechanically diverse compounds.