Responses to various stressors in the brain change with age. However, little is known about the neural mechanisms underlying age-dependent changes in stress responses. It is known that serotonin, a stress-related transmitter, is closely related with the regulation of stress responses in the brain and that serotonergic function is modulated by various factors, including estrogen, in both sexes. In the present study, to elucidate the effects of aging on stress responses in serotonergic neurons, we examined the expression levels of tryptophan hydroxylase (TPH; a marker of serotonergic neurons) in the dorsal, ventral and lateral parts of the dorsal raphe nucleus (DRN) in young and old intact male rats. In young males, repeated restraint stress significantly increased the number of TPH-positive cells in all subdivisions of the DRN. In contrast, the stress-induced increase in TPH expression was only observed in the ventral part of the DRN in old males. Pretreatment with an estrogen receptor β antagonist had no effect on the number of TPH-positive cells in the dorsal and lateral DRN in young stressed males, whereas the antagonist decreased the number of TPH-positive cells in all DRN subdivisions in old stressed males. Our results suggest that the effects of repeated stress exposure on the expression of TPH in serotonergic neurons in the DRN change with age and that estrogenic effects via estrogen receptor β on TPH expression in stressed old males differ from those in young males.