Infant guinea pigs, 11/12 and 18/19 days of age, exhibited high rates of vocalizing and increases in plasma concentrations of cortisol when separated from their mothers for 30 min in a novel environment (unfamiliar cage in an unfamiliar room). Levels of vocalizations and plasma cortisol were significantly reduced when the pup was exposed to the novel environment with its own conscious or anesthetized mother, but not when it was exposed with an unfamiliar anesthetized lactating female. Pups spent equivalent amounts of time in physical contact with their own conscious mother, their own anesthetized mother and an unfamiliar anesthetized lactating female. Unexpectedly, when the unfamiliar cage was placed in an environmental chamber during separation, pups showed negligible increases in vocalizations and plasma cortisol levels, even when administered footshock just prior to exposure. These findings demonstrate that guinea pig pups exhibit adrenocorticoid as well as behavioral responses to maternal separation, provide further evidence for the existence of filial attachment in this species, and document an unexpected role of the test environment in determining responses during maternal separation. The guinea pig may afford a rodent model for examining the effects of attachment-object separation on the activity of the pituitary-adrenal system.