ObjectiveTo determine brain dialysate and serum melatonin (MLT) levels in adult rats by high-performance liquid chromatography (HPLC) with fluorescence detection and enzyme-linked immunosorbent assay (ELISA), and compare the sensitivity, accuracy and reliability of the two detection methods.MethodsThe guide tube was implanted into the striatum of healthy adult rats.One week after implantation, dialysate samples were collected at 2:00, 6:00, 10:00, 14:00, 18:00, 22:00 h in striatum by brain microdialysis.Blood samples were collected from the rat tail vein at 22:00 h on the following day.HPLC with fluorescence detection and ELISA were applied to detect dialysate and serum MLT levels.ResultsThe lowest concentration of MLT that can be detected by HPLC with fluorescence detection was 0.1 pg/mL.There was a regressive equation y=10 446 x+8 603.5 (r=0.998 7, P<0.05) in linear range 1.0~500 pg/mL of MLT levels, with an intra-assay and inter-assay of below 6%.The detection limit of MLT by ELISA was 5.92 pg/mL, and the detection range was 12.35~1 000 pg/mL.The mean concentrations of dialysate MLT that showed a distinct fluctuation were (in pg/mL) （32.99±2.22）, （18.25±2.27）, （4.03±1.70）, （2.92±1.08）, （12.08±1.77）, （21.83±2.04） at 2:00, 6:00, 10:00, 14:00, 18:00 and 22:00 h by HPLC with fluorescence detection.The recovery of the probes was from 12% to 13%.Besides, the mean concentration of serum MLT was （82.62±8.09） pg/mL by this method.The extraction recovery of serum MLT was more than 90%.There was a distinct positive correlation between serum MLT and dialysate MLT levels at 22:00 pm, and pearson’s correlation coefficient between the two was 0.987 at the 0.001 level (two-tailed). In addition, the mean concentration of serum MLT was （81.31±9.62） pg/mL by ELISA.There was no significant difference in serum MLT levels between the two methods (P=0.115).ConclusionThese data suggest that HPLC with fluorescence detection may be sensitive, rapid, accurate and reproducible in determination for MLT levels in dialysate and serum samples, and serum MLT levels by HPLC with fluorescence detection may be consistent with those by ELISA.