Infant squirrel monkeys had their mother removed from the home cage for 2 hr on 80 occasions between 12 and 31 weeks of age. The initial separation elicited high levels of infant vocalizations. By the second observation session (Separation 14), no vocal response to separation was observed. Plasma cortisol levels were markedly elevated at the beginning of the separation series, and there was a significant decline in this response by Separation 28, which was the next separation during which cortisol was monitored. However, there was no further decline, so that significant cortisol elevations were observed throughout the remainder of the separation series. At 35 weeks, the infants were separated one additional time and their responses were compared to those of a previously nonseparated control group. The control group vocalized more, but the two groups exhibited equivalent cortisol elevations. Observations of time spent riding on the mother during undisturbed conditions indicated that both groups developed independence from the mother at about the normal rate. Overall, the data show that brief separation from the mother can activate the infants' pituitary-adrenal system even when the infant has been separated 80 times previously, no longer appears behaviorally responsive to separation, is almost 9 months of age, and exhibits normal signs of independence from the mother.