The flavonolignan silybin (1), isolated from the seeds of milk thistle (Silybum marianum), occurs in nature as an equimolar mixture of two diastereoisomers, silybin A and silybin B, that exhibit different biological activities. The preparative production of optically pure silybin A and B in a diastereoisomeric purity greater than 95% was accomplished using immobilized Candida antarctica lipase B (Novozym 435) in a combination of two reactions: regioselective acetylation of a natural silybin mixture (1) and subsequent stereoselective alcoholysis of the resulting 23-O-acetylsilybin (2). Several grams of the optically pure substances can be produced within one week using this new, robust and scalable process, which is selective, mild and high-yielding. (C) 2010 Elsevier Ltd. All rights reserved.