The effects of native cyclodextrins (α, β or γ), hydroxypropyl-β-cyclodextrin, β-cyclodextrin solubilized in urea, soluble starch and glucose in water solution on the fluorescence behaviour of melatonin (N-acetyl-5-methoxytryptamine) (M) and 5-methoxytryptamine [5-methoxy-3-(2-aminoethyl)indole] (5M) were determined. In addition, the effect of methanol and propanol with and without β-cyclodextrin or hydroxypropyl-β-cyclodextrin was assessed. From the fluorescence changes with pH, the values of the pKa for the ground (9.9 ± 0.2) and the excited state (7.7 ± 0.2) for 5M were determined. From the fluorescence changes with β-cyclodextrin or hydroxypropyl-β-cyclodextrin, the association constants of M, 5MH [5-methoxy-3-(2-ammoniumethyl)indole] and 5M with the two hosts were determined. The values with β-cyclodextrin were KAssoc5MH = (1.4 ± 0.4) × 102 mol−1 dm3, KAssoc5M = (1.6 ± 0.1) × 102 mol−1 dm3 and KAssocM = (1.1 ± 0.2) × 102 mol−1 dm3, and with hydroxypropyl-β-cyclodextrin KAssoc5MH = (1.1 ± 0.3) × 102 mol−1 dm3, KAssoc5M =(2.5 ± 0.1) × 102 mol−1 dm3 and KAssocM = (1.51 ± 0.07) × 102 mol−1 dm3. The ratios of the fluorescence quantum yields for the bound and free substrate (phisb/phisf) were in the range 1.15–1.48. The detection limits under the optimum conditions were 0.381 ± 0.001 ng cm−3 for the complex 5MH–hydroxypropyl-β-cyclodextrin in water and 0.290 ± 0.001 ng cm−3 for the complex M–hydroxypropyl-β-cyclodextrin in water with 5% of methanol. The recovery of melatonin from pharmaceutical preparations was 98–103% with an RSD of 2%. The recovery from rat pineals was also good. The method is direct, simple and accurate.