This study investigated the effects of neonatal handling and maternal separation on the development of anxiety behavior and the hypothalamic-pituitary-adrenal axis of C57BL/6 mice. We hypothesized short periods of neonatal handling would diminish anxiety and secretion of corticosterone, while longer periods of maternal separation would elevate anxiety and plasma corticosterone compared to a nonhandled group. Mice were bred and reared as follows. After birth, each litter was assigned to one of four groups: mother and pups removed from the home cage for 10 min (group 1) or 180 min a day (group 2); mother only removed from home cage 180 min a day (group 3); and no handling until weaning (group 4). All separation occurred on the first 10 days of life. Juvenile males that experienced 10 min of separation/day exhibited decreased anxiety behavior compared to all other mice. A second group of litters were bred and reared according to groups 1, 2, and 4 as described above. Upon adulthood, anxiety behavior was assessed in males, and the corticosterone response to an acoustic stressor was quantified. No effect of differential rearing was observed on behavior, but there was a marked effect on plasma corticosterone secretion between the groups. Adult male mice neonatally handled for 10 min/day exhibited a blunted corticosterone response, and mice that experienced 180 min of maternal separation exhibited a prolonged corticosterone response to the acoustic stimulus compared to the nonhandled group. These results demonstrate the development of the mouse's hypothalamic-pituitary-adrenal axis can be modified by neonatal rearing conditions, and suggest that the mouse could be a viable animal model to determine the genetic-environmental interactions governing brain development.