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The determination of melatonin has been reported in several fruits, including cherries. This study had the goal to evaluate melatonin levels present in five different cherry cultivars from the region of Fundao in Portugal, a well-known location for this fruit cultivation. Three of the five cultivars are evaluated for the first time regarding melatonin levels (Saco, Summit and Sunburst). To determine the presence of this hormone, a reliable analytical method was developed and fully validated, and an SPE procedure was applied and optimized using Oasis (R) HLB cartridges to concentrate melatonin and clean-up the samples before analysis by high-performance liquid chromatography coupled to electrochemical detection. The extraction efficiency from fresh matrices varied from 74 to 91%, while the absolute recoveries (SPE clean-up) of melatonin ranged from 61 to 75%. The procedure was considered linear for concentrations ranging from 0.025 to 4 mu g/mL with a mean R-2 value of 0.9974 and with calibrators’ accuracy (mean relative error) within a +/- 15% interval for all concentrations. The concentrations of melatonin found in the different cherry cultivars ranged from 11 to 28 ng/g of fresh fruit, and Burlat cultivar was the one presenting the highest level of this hormone.