Propolis is used by bees for strengthening and waterproofing a hive, and for sterilizing the hive against microbial infections. Propolis contains a high concentration of flavonoids, which are used in a wide range of cosmetic and health food preparations for their antimicrobial properties. Propolis is usually dissolved in ethanol or ethanol/water mixtures to remove insoluble material such as waxes and detritus from the hive. The resultant solution is a propolis tincture. A new supercritical antisolvent/extraction process has been developed for the fractionation of propolis tincture to obtain flavonoids and essential oil fractions by extraction, and remove high molecular mass components by antisolvent precipitation. Flavonoids are practically insoluble in pure CO2, but sufficiently soluble in CO2 + ethanol to enable their separation from high molecular mass and/or more polar components. In the first step of the process, supercritical CO2 is Used both as an anti-solvent to precipitate high molecular mass components, and as a solvent to extract the ethanol and soluble components of the propolis. This extract is then fractionated in two separation steps to create a concentrated flavonoid fraction as the primary product, and an essential oil/ethanol fraction as a secondary product. The effects of pressure, temperature, flow rate ratio, tincture composition and tincture concentration on product quality and yield were determined at a laboratory and pilot scale. The tincture concentration of propolis has the greatest effect on the yield and concentration of flavonoids in the product fraction when pure ethanol is used as the solvent. The flow rate ratio becomes important when the tincture also contains water. The process has been successfully scaled up to a demonstration scale using optimized pressure, temperature, flow ratio and tincture concentrations obtained from laboratory and pilot scale trials. (C) 2003 Elsevier B.V.. All rights reserved.