Abstract: Hairy root culture of the medicinal plant Rheum palmatum L. was established by genetic transformation with Agrobacterium rhizogenes. The effects of various media with different pH on growth of the hairy roots and biosynthesis of free anthraquinones were investigated. The experimental results showed that MS agar medium with pH 5.5～5.8 is suitable for growth of the hairy roots. Dark condition is favourable and 62.5-fold increase in fresh weight was reached within a culture period of 25 days. Auxin (0.1 mg·L^-1 IAA) activated the hairy root growth but inhibited the biosynthesis of free anthraquinones. About 28% of the total free anthraquinones was released into the liquid medium from the rhubarb hairy roots.