Preweaning guinea pigs were placed into a test cage either alone or with their mother for 10, 30, or 90 min, or were placed into the cage alone for 24 hr. At 30 and 90 min, pups exhibited higher plasma levels of ACTH if alone than if with the mother. This effect was not apparent at 10 min, and ACTH levels of pups tested alone were no longer greater than baseline levels at 24 hr. These results provide further evidence that brief maternal separation can serve as a potent stimulus for activation of the hypothalamo-pituitary-adrenal system in this species. Plasma epinephrine and norepinephrine concentrations of pups tested alone were not significantly greater than those of pups tested with their mothers. But, pups tested alone for 10 and 30 min did show an elevation of epinephrine and norepinephrine over baseline levels. That is, the combined influence of separation and other aspects of the manipulation (e.g., exposure to novelty) evoked sympathetic responses. In addition, the number of vocalizations emitted by pups during the first 30 min of isolation in the test cage was positively correlated with concentrations of ACTH, epinephrine, and norepinephrine following 24 hr of this procedure, but not following 90 min or at baseline. Thus, the initial behavioral response appears to be predictive of the levels of both hypothalamo-pituitary-adrenal and sympathetic activity 24 hr after separation is initiated.