Using two specific anti-peptide antibodies (WH-66 and WH-412) against tryptophan hydroxylase (TPH or WH), a single principle band from the midbrain raphe area was seen (approximately 49 kDa) in immunoblots. Densitometric comparison of the immunoreactivity of the 49 kDa band was greater (50–75%) in immunoblots of midbrain raphe samples from adrenalectomized (ADX) rats given dexamethasone (DEX) in their drinking water (10 mg/liter) for 12–96 hr. No difference from ADX brains was seen in the 49 kDa band after only 4 hr of exposure to DEX in the drinking water. Immunocytochemical staining with WH-66 of sections from rat brainstem showed specific cellular staining in all of the serotonergic raphe nuclei but not in substantia nigra or locus coeruleus. More cellular staining of WH-66-immunoreactive (WH-66-IR) cells was observed in the dorsal and median raphe nuclei in ADX rats given DEX for 72 hr, especially in the perikarya and in the primary dendrites. Quantification of staining per cell soma with an automatic image analyzer indicates that amount of WH-66-IR in neurons from both the lateral wing subdivision of the dorsal raphe nucleus and in the supralemniscal nucleus, B-9, was 80% higher in the ADX+DEX compared to ADX animals. Interestingly, morphometric analysis of these same cells showed a corresponding increase of 37–80% in somal area. It is suggested that a part of the increase in TPH/WH staining may be a consequence of cellular hypertrophy due to DEX treatment of the ADX rats.