Objective To study the effect of ethonal on the serotonergic systems by analyzing the altered expression of tryptophan hydroxylase (TPH), serotonin (5-HT) and serotonin transporter (SERT) in the brain of ethanoltreated rats. Methods Wistar rats were treated with 20% ethanol for 6 months. Immunocytochemistry, flow cytometry and Western blotting were used to analyze the altered expression of the serotonergic markers in different brain regions of the ethanoltreated rats. Results 1. Immunohistochemistry showed the number of TPH or 5-HT positive neurons in dorsal raphe nucleus (DRN) is less in ethanoltreated rats than that in control (EM>P/EM>0.01), the diameter of TPH neurons in DRN is smaller than that in control (EM>P/EM>0.01) and the mean gray value of TPH, 5-HT and SERT in observed regions of the ethanoltreated rats is larger than that in control (EM>P/EM>0.05). 2. Flow cytometry showed the expression of TPH, 5-HT and SERT in the studied regions of the ethanoltreated rats decreased significantly compared with control (EM>P/EM>0.05). 3. Western blotting showed the ratio of SERT/β-actinin or TPH/β-actinin in analyzed regions of the ethanol-treated