In order to approach lucidin formation (a strong mutagen or a carcinogen) from a physiological standpoint, hairy roots of Rubia tinctorum L. were established by a transformation of Agrobacterium rhizogenes strain 15834 and cultured in a liquid woody plant medium without plant hormones. The anthraquinone pigment composition of the intact hairy roots was essentially the same as that of the intact non-transformed (normal) roots, in which lucidin O-beta-D-primeveroside (LuP) was one of the major pigments. Lucidin was scarcely detected in the intact hairy roots, but was a main pigment after the squash treatment. The crude protein extract of intact hairy roots exhibited LuP-glycosidase activity (an activity converting LuP to lucidin). This activity was also detected in the roots of the normal plants at a high level, but slightly in the stems and not in the leaves. Methyl jasmonate enhanced the LuP production and LuP-glycosidase activity in the hairy roots. On the other hand, ethephon or salicylic acid had either no effect or rather an inhibitory effect on them. After partial purification of LuP glycosidase, the resultant active fraction producing a major band with an apparent Mr of 68 kDa exhibited the substrate specificity for both aglycon and sugar-moiety. The sugar released from LuP by this fraction was neither D-glucose nor D-xylose and was hydrolyzed into them. These results suggest that LuP specific beta-primeverosidase (EC 3.2.1.149) exists in the roots of R. tinctorum and is involved in the systematic defense system.