Hyoscyamine-6beta-hydroxylase (H6H; EC 1.14.11.11) catalyses oxidative reactions in the biosynthetic pathway leading from hyoscyamine to the more pharmaceutically valuable tropane alkaloid scopolamine. The h6h gene encoding H6H from Hyoscyamus niger was introduced, under the control of the CaMV 35S promoter, into non-hyoscyamine-producing Nicotiana tabacum and hyoscyamine-producing Hyoscyamus muticus. The transformation was performed using a binary vector system based on Agrobacterium rhizogenes. Production of scopolamine in hairy roots was clearly correlated with the 35S-h6h transcript expression. The engineered N. tabacum and H. muticus hairy roots were studied for the production of scopolamine and other tropane and nicotine alkaloids after feeding the cultures with exogenous hyoscyamine. N. tabacum hairy roots carrying the 35S-h6h transgene showed a more efficient uptake of hyoscyamine from the culture medium and a higher rate of bioconversion of hyoscyamine to scopolamine than those of H. muticus. In particular, the secretion of scopolamine in N. tabacum hairy roots was remarkably high, up to 85% of the total scopolamine being released to the culture medium. Exogenous hyoscyamine also caused changes in nicotine alkaloid accumulation in N. tabacum hairy roots.