Both the magnitude and duration of the hormonal stress response change dramatically during neonatal development and aging, as well as with prior experience with a stressor. However, surprisingly little is known in regards to how pubertal maturation and experience with stress interact to affect hypothalamic-pituitary-adrenal (HPA) axis responsiveness. As adolescence is a period of neurodevelopmental vulnerabilities and opportunities that may be especially sensitive to stress, it is imperative to more fully understand these interactions. Thus, we examined hormonal and neural responses in prepubertal (28 days of age) and adult (77 days of age) male rats after exposure to acute (30 min) or more chronic (30 min/day for 7 days) restraint stress. We report here that after acute stress, prepubertal males exhibit a significantly prolonged hormonal stress response (e.g. adrenocorticotropic hormone and total and free corticosterone) compared with adults. In contrast, after chronic stress, prepubertal males exhibit a higher response immediately after the stressor, but a faster return to baseline, compared with adults. Additionally, we demonstrate that this differential stress reactivity is associated with differential neuronal activation in the paraventricular nucleus of the hypothalamus (PVN), as measured by FOS immunohistochemistry. Using triple-label immunofluorescence histochemistry, we found that a larger proportion of CRH (CRH), but not arginine vasopressin (AVP), cells are activated in the PVN in response to both acute and chronic stress in prepubertal animals compared with adults. These data indicate that experience-dependent plasticity of the HPA neuroendocrine axis is significantly influenced by pubertal maturation.