The application of supercritical carbon dioxide in the extraction of bioactive compounds from three varieties of Helianthus annuus L. (Stella, Arianna and Hilian) has been investigated. The influence of different variables, including pre-treatment of the sample, temperature and pressure, was analyzed. The best extraction yields and activity profiles were obtained for the Arianna variety. Several sets of conditions for fractionations using two cyclonic separators were studied and the bioactivities and extraction yields of the fractions were compared. The best fractionation conditions were obtained at 90 bar and 40 degrees C. During the fractionation process it is possible to obtain an extract that is free of chlorophylls, a situation that makes the purification process and the identification of secondary metabolites easier. (C) 2011 Elsevier B.V. All rights reserved.