BACKGROUND: Hyperoside is a valuable natural pharmaceutical that has many biomedical functions. By the traditional method of alcohol extraction, only hyperoside solution of very low concentration can be obtained, so a new method is urgently needed to produce pure hyperoside more effectively. Reverse micellar extraction has been widely used in the purification of biological macromolecules. Theoretically, this method could also be used to purify materials with small molecules. Therefore it would seem appropriate to consider the extraction of hyperoside, a material with small molecules, using reverse micelles. In this study the factors affecting hyperoside extraction using cetyl trimethylammonium bromide (CTAB) reverse micelles were comprehensively investigated. RESULTS: The results showed that the partition coefficient increases with an increase in aqueous phase pH or CTAB concentration before reaching a saturated state. The existence of anions in the system can affect the extraction in several ways. For example, SO42- and PO43- ions can form hydrogen bonds with hyperoside and thereby improve the extraction significantly. CONCLUSION: This study proved that extraction using CTAB reverse micelles is a very effective method to separate hyperoside from Hypericum perforatum L. Under optimal conditions the extraction efficiency can reach 68.3% and the single-strip-extraction efficiency can reach 85.9%. The proportion of hyperoside among the flavonoids left in the strip-extract can reach 93.3 wt%. This finding is very significant, because it demonstrates that reverse micellar extraction is a promising way to separate and purify materials with, small molecules. (c) 2008 Society of Chemical Industry.