The influence of different parameters on the Supercritical Carbon Dioxide (SCO2) extraction of Italian Propolis were studied with attention to extraction yield and chemical composition of obtained fractions. Operating parameters of SCO2 were optimized using central composite design. Analysis by multiple regression indicated that pressure and time have a major linear effect on the extraction yield and extract composition. Propolis SCO2 extracts present different chemical content compared to the ethanolic extract obtained with ultrasound assisted extraction (UAE). SCO2 extract can be used to perform further UAE ethanol extraction obtaining a flavonoid containing extract. These findings indicate two possible applications of supercritical carbon dioxide for Propolis extraction, one to obtain lipophilic fractions enriched in specific constituents, the other as pre-treatment of the crude material to facilitate the further extraction with ethanol. (C) 2014 Elsevier B.V. All rights reserved.