Chronic stress and alterations in the serotonergic system are key predisposing factors to the development of major depression. Tryptophan hydroxylase (TPH) is the key enzyme in the biosynthesis of serotonin (5-HT). The effects of chronic stress on TPH activity remain uncertain. The BALB/c strain is stress-sensitive, highly anxious and possess a single nucleotide polymorphism in their tryptophan hydroxylase (TPH) 2 gene (tph2), resulting in reduced levels of central serotonin compared to C57BL/6J mice, which harbour the wild-type allele. We examined the effects of repeated restraint stress on the serotonergic system and TPH activity in these two inbred strains. TPH activity was assessed by accumulation of 5-hydroxytryptophan, a rapidly decarboxylated intermediate metabolite of tryptophan and precursor of 5-HT, using an enzyme inhibition strategy. Furthermore, the concentrations of 5-HT and its major metabolite 5-hydroxy indole acetic acid were assessed. Interestingly, 5-HT turnover was significantly increased in the majority of the brain regions assessed following acute stress in C57BL/6J. In contrast, BALB/c mice exhibit significant increases in 5-HT turnover in the striatum and hippocampus only following repeated stress. On the other hand, TPH activity was significantly decreased in the brainstem and cortical regions of C57BL/6J mice following both acute and chronic stress. Conversely, no significant stress-induced change in BALB/c TPH activity was observed. Together these data highlight the differential serotonergic response of BALB/c and C57BL/6J mice to acute and chronic restraint stress and may offer insight into the observed differences in their stress-related phenotypes.