The UV-suppressive effect of topical melatonin was assessed at different application time points in a double-blind randomized clinical trial. The lower back of 20 healthy volunteers was treated with 0.6 mg/cm2 melatonin or vehicle either 15 min before or 1, 30 or 240 min after UV irradiation. The erythema was evaluated visually and measured by chromametry 24 h after irradiation. UV-absorbing effects of melatonin were measured at a concentration of 8 μg/ml with a spectrophotometer. Melatonin absorbs UV light at a wavelength of 225–275 nm which is clearly below the wavelength of UVA and UVB (290–390 nm). The visual score showed that application of melatonin 15 min before irradiation significantly suppressed erythema compared to treatment with vehicle alone (p < 0.001). Similar results were found by chromametry (p < 0.001). Treatment after irradiation showed no UV suppression. The erythema suppressive effect of melatonin might be explained by the radical-scavenging mechanism of quenching meanly hydroxyl radicals (.OH) which are known to be most present in sunburn reaction of the skin. The protective effect of the pre-irradiation treatment might be explained by penetration into the skin within 15 min and the presence in a local concentration at the irradiation time point.