This study was conducted to examine the pathophysiologic mechanisms of long-term adverse effects by neonatal maternal separation on neurobehaviors of the offspring. Sprague-Dawley pups were separated from dam daily for 180min during the first 2 weeks of life (MS) or undisturbed (NH), and subjected to behavioral sessions for ambulatory activity, forced swim, and elevated plus maze tests at 2 months of age. Serotonin reuptake transporter (5-HTT) mRNA levels in the raphe nucleus and the contents of serotonin (5-HT) and its metabolite 5-hydroxyindol acetic acid in the raphe and the hippocampus were examined as well. Ambulatory counts decreased and immobility duration in swim test increased in MS rats compared with NH rats. MS rats spent more time in the closed arms, less time in the open arms, of elevated plus maze, compared to NH rats. The hippocampal contents of 5-HT and the raphe expression of 5-HTT mRNA were decreased in MS rats compared with NH rats. These results suggest that neonatal maternal separation may result in the development of depression- and/or anxiety-like behaviors in later life, in which the long-term alterations in 5-HTergic neurotransmission may take a role.