Neonatal handling permanently alters hypothalamic-pituitary-adrenal responses to stress. This effect is, in part, mediated by a handling-induced increase in forebrain glucocorticoid receptor gene expression. The effect of postnatal handling on glucocorticoid receptor expression appears to be mediated by an increase in serotonin (5-HT) activity, acting via a 5-HT2 receptor with a high affinity for 5-HT (i.e. the 5-HT2H receptor). In the present study we examined the nature of the effects of handling on the relevant 5-HT systems. We found that: (1) handling increases 5-HT turnover in regions of the neonatal rat brain where glucocorticoid receptor expression is altered (i.e. the hippocampus and frontal cortex), but not in regions where glucocorticoid receptor expression in unaffected (e.g. hypothalamus and amygdala); (2) handling has no long-term effects on hippocampal or frontal cortex 5-HT turnover, and is actually associated with a decrease in 5-HT concentrations; and (3) handling does not alter 5-HT2 receptor density in the hippocampus or frontal cortex in neonates (although there are surprising effects on 5-HT2 receptor density in the frontal cortex of adult animals). Taken together these data provide further evidence for the importance of 5-HT in mediating the effects of handling on the development of glucocorticoid receptor expression, but suggest that the role of 5-HT is unique to early development; differences in glucocorticoid receptor expression in adult handled and non-handled animals are not associated with long-term differences in either 5-HT levels or 5-HT2 receptors.