The effects of 30-min separation from peers on behavior and plasma cortisol levels were studied in juvenile, mother-reared, female squirrel monkeys, housed in triads. Juveniles vocalized much more frequently when isolated in a novel room than they did when either undisturbed, handled only, placed with both peers in a novel room, or separated from one peer and housed with the other in the home cage. When separated from one peer in the home cage, the monkeys engaged in putative scent-marking behaviors more frequently than when undisturbed. Exposure to the novel room resulted in increased vocalizing, and decreased locomotor activity and scent marking, regardless of whether the subject was isolated or with peers. Plasma cortisol levels were elevated following all manipulations and no effects could be attributed specifically to separation or exposure to novelty. These results demonstrate that brief separation from peers can evoke a behavioral reaction in juvenile squirrel monkeys that resembles the behavioral “protest” response of maternally-separated infants, and that the response of juveniles, like that of infants, varies greatly with the separation procedure employed.