Methylphenidate (MP) is widely used to treat attention deficit/hyperactivity disorder in children. However, basic research has been mainly focused on MP treatment in adult, behaviorally normal rodents. Here we analyzed MP-evoked changes of dopamine (DA) release in the limbic system of juvenile rodents with hyperactive and attention deficit-like symptoms. Using dual probe in vivo microdialysis, DA levels were quantified in the medial prefrontal cortex and nucleus accumbens of juvenile and adolescent degus (Octodon degus). Acute stress- and acute MP-evoked dopaminergic responses in normal juvenile and adolescent animals were compared with (i) animals showing symptoms of hyperactivity and attention deficits induced by early life stress, i.e. repeated parental separation during the first 3 weeks of life, and (ii) animals chronically treated with MP during pre-adolescence. Our main results revealed that (i) early life stress and (ii) chronic MP treatment during pre-adolescence cross-sensitize limbic dopaminergic functions in adolescent animals. Furthermore, we demonstrated a unique pattern of acute MP-evoked DA release in the juvenile compared with the adolescent medial prefrontal cortex and nucleus accumbens. Our findings that the functional maturation of dopaminergic limbic function is significantly altered by early life experience, i.e. repeated parental separation and chronic MP treatment, allow novel insights into the etiology of attention deficit/hyperactivity disorder and into the long-term consequences of MP treatment on brain development.