In this study we addressed whether certain behavioural measures, endocrine levels and specific stress-related proteins exhibit long-term alterations in adult rats following repeated postnatal maternal separation. Rats were subjected to daily maternal separation for 15 min (HMS15) or 180 min (HMS180) from postnatal day 2–14. Adult HMS180 animals were hypoactive and had increased levels of stereotypy compared to HMS15 and normal animal facility-reared (AFR) animals. HMS180 animals also had augmented plasma adrenocorticotropin (ACTH) and corticosterone (CORT) concentrations following an acute stressor, compared to the other two groups. We assessed persistent changes in proteins regulated by stress in hippocampus, cortex, ventral tegmental area, nucleus accumbens, striatum and amygdala. Western blotting analysis revealed a decrease in the levels of mature brain-derived neurotrophic factor (BDNF) in hippocampus and striatum, but an increase in the ventral tegmental area in the HMS180 rats. Levels of pro-BDNF were significantly increased in the ventral tegmental area of HMS180 animals but were unchanged in other brain regions compared to the other two groups. Levels of the transcription factors cAMP response element binding protein (CREB) and ΔFosB were unchanged in all of the brain regions studied in the maternally separated rats. These data show that maternal separation induces long-term changes in BDNF expression, and more specifically the processing of BDNF, in the hippocampus, striatum and ventral tegmental area. Recognition of these adaptations begins to define the brain regions, and neural circuitry, associated with persistent alterations induced by early life stressors and the development of mood disorders.