Hairy root cultures of Centranthus ruber DC. were established by infection of sterile plantlets with Agrobacterium rhizogenes, strain RI601. The transformed roots were grown in 12 different, hormone-free liquid media, and valtrate, isovaltrate, 7-desisovaleroyl-7-acetylvaltrate, 7-homovaltrate, didrovaltrate and isovaleroxyhydroxydidrovaltrate were quantified by high performance liquid chromatography. The highest overall valepotriate content (3.0% dry wt) was observed in half-strength Gamborg B5 medium supplemented with 3% sucrose. This concentration is very similar to that found in the roots of parent plants grown in the field. The use of N,N-dimethylmorpholinium iodide, a plant bioregulator, was very detrimental to the hairy root growth and to the valepotriate production. The hairy roots cultured in haft strength Gamborg B5 liquid medium supplemented with 3% sucrose for 45 days produced over 31 mg/g dry wt valepotriates.