Hairy root cultures of Valeriana officinalis var. sambucifolia were established by infection of sterile plantlets with Agrobacterium rhizogenes strain R1601.The transformed roots were grown in 10 different, hormone-free liquid media and the isovaltrate, valtrate, didrovaltrate, isovaleroxyhydroxydidrovaltrate content was quantified by HPLC. Valepotriates were entirely retained inside the root tissues. The highest overall valepotriate content (10.3 % dry wt), 4 times the amount found in the roots of 9-month-old nontransformed plants, was observed in half strength Gamborg B5 medium supplemented with 2 % sucrose. The hairy roots cultured in Murashige and Skoog liquid medium supplemented with 2 % sucrose for 50 days produced over 44 mg/g dry wt valepotriates.