Silymarin production by HAIRY ROOT culture of Milk thistle (Silybum marianum L. Gaertn) was investigated using Agrobacterium rhizogenes AR15834. HAIRY ROOTs were induced by injection or inoculation of explants with A. rhizogenes. One month old HAIRY ROOTs were dissected from the explants and grown in Murashing and Skoog (MS) liquid medium. Polymerase Chain Reaction (PCR) using the bgene and the b-glucoronidase (GUS) assays were used for identification of the transformed HAIRY ROOTs. Flavonolignan levels in the HAIRY ROOTs were determined by high-performance liquid chromatography (HPLC). Five different components were isolated; taxifolin, silychristin, silydianin, silybin and isosilybin, with the following quantities, 0.009, 0.041, 0.042, 0.007 and 0.011 mg g-1 dry weihht, respectively. Silybin was the major flavonolignan. Produced by HAIRY ROOTs culture may serve as a useful system for producing silymarin or studying its biosynthetic pathways.