The behavioral and hormonal responses of mother and infant squirrel monkeys (Saimiri sciureus) were examined to assess temporal and environmental factors that influence the response to separation. In two experiments evaluating the effects of 1-, 3-, 6-, and 24-hr separations, it was found that signs of infant behavioral agitation decreased over time, whereas adrenocortical activation persisted or even increased. Moreover, two separation environments were shown to differentially affect behavioral and hormonal responses. Separated infants vocalized significantly more when their mothers were proximal than when isolated, but showed lower cortisol levels in the adjacent separation than in the total-isolation condition. These data indicate that the intensity of the infant's calling response cannot be used to predict internal state (as reflected by cortisol levels). Furthermore, vocalization rate is highly dependent upon contingent stimuli, such as the presence of maternal cues. Following separation, the mothers also showed elevated cortisol levels. However, both the magnitude and pattern of the response differed considerably from that of the infant.