Eugenia uniflora L. is commonly known as “pitanga” in Brazil and belongs to the family Myrtaceae. This species is a plant with well-known edible fruits, which flavor has economic added value for the perfume industry, being widely used in cosmetics, soaps and shampoos. Literature sources show many works describing the volatile profile of the E. uniflora varieties, however no separation procedures were found to obtain the essential oil metabolites of this plant1. The aim of this work was to promote the separation and chemical analysis of major volatile metabolites present in the essential oil of Eugenia uniflora L. The plant leaves (100.0g) were collected on the campus of Oswaldo Cruz Foundation (FIOCRUZ) and were submitted to hydrodistillation for two hours in a modified Clevenger apparatus. The leaf essential oil was analyzed by GC-FID and GC-MS. The oil obtained (1.0g) was subjected to countercurrent chromatography for the isolation of its major components, such as the sesquiterpenes (±)-selin-1,3,7(11)-trien-8-one and (±)-oxidoselin-1,3,7(11)-trien-8-one. These metabolites account for 32.7% and 24.5% of the 53 components present in the mixture, respectively. The HSCCC has been used for isolation of many biologically active compounds from plant extracts with high percentage of sample mass recovery, fast separation process and for the smaller solvent volumes required2. Among the tested solvent systems suitable for low polarity compounds, the hexane/acetonitrile 1:1 (v/v) was chosen for the separation process. An isocratic elution mode was chosen for the separation process of 1.0g of the crude essential oil. A total of 120 fractions were collected, 80 of them obtained with rotation of 860 rpm and the last 40 obtained with rotation turned off in each separation running. The obtained fractions were analyzed after thin-layer chromatography and by GC-FID and GC-MS. In the first chromatographic separation, acetonitrile was used as stationary phase. The obtained fractions 72-80 (70.0mg) showed a content of (±)-oxidoselin-1,3,7(11)-trien-8-one in purity level superior to 96% by GC-FID. When hexane was used as the stationary phase it was possible to obtain the fractions 23-29 (55.0mg) with content of (±)-oxidoselin-1,3,7(11)-trien-8-one ranging from 90-95% purity, as well as fractions 34-35 (24.0mg) which were found to contain (±)-selin-1,3,7 (11) -trien-8-one in 90.0% purity by GC-FID. The isolated metabolites were analyzed by NMR and sent to biological assays. The countercurrent chromatography technique was efficient in the separation of sesquiterpenes of structure and polarity very similar in a short time, with great solvent economy and with high purity. Acknowledgments: We thank Farmanguinhos/Fiocruz for financial support.