Neonatal maternal separation has been widely used to model the well-established causal relationship between stress in early life and the later development of depression. As corticotrophin-releasing factor (CRF) and vasopressin (AVP) have been implicated in depression, we aimed to determine the long-term effects of maternal separation stress on these neuropeptide systems, and also to explore whether these effects are gender-dependent. Immunohistochemical staining of CRF, AVP and c-Fos was used to assess whether these neuropeptide systems were affected following an acute swim stress in male and female maternally separated rats. There was an increase in CRF-immunoreactivity (IR) (p<0.05), and an increased co-localisation of c-Fos and CRF (p<0.05) following stress, in the paraventricular nucleus of the hypothalamus (PVN) of maternally separated female rats only. We found no differences in CRF in the hypothalamus of maternally separated and control male rats. However, male maternally separated rats exhibited decreases in AVP-IR in both the non-stressed and stressed groups relative to controls (p<0.001). These data provide further evidence of the involvement of the neuropeptides CRF and AVP in the long-term maladaptive effects of maternal separation stress in early life. The enhanced CRF response to stress in MS females relative to males suggests that maternal separation stress results in a more reactive neuroendocrinological stress system in females, than in males. Furthermore, the sexually dimorphic effects of maternal separation on these neuropeptides indicate that gender is an important factor influencing the trajectory of early life stress effects on CRF and AVP systems in the brain.