Two series of Atropa belladonna hairy root lines were obtained, the first one transformed via Agrobacterium tumefaciens harboring rol C and npt II genes, and the other transformed with rol ABC and npt II genes. Thirteen hairy root lines were obtained and selected on hormone-free medium. The transformation was confirmed by PCR analysis, and these root lines were first examinated for their growth rate. Then hyoscyamine and scopolamine production was measured after 3 and 4 weeks of culture to evaluate the possible role of rol C gene in tropane alkaloid formation. The rol C gene alone played a significant role in the hairy root growth rate (17-fold increase). However this effect was much lower than that induced by the rol ABC genes together (75-fold increase). In contrast, the rol C gene alone was as efficient as the rol ABC genes together (mean value of total alkaloids: 0.36% dry weight, i.e., 12-fold times more than in untransformed roots) to stimulate the biosynthesis of tropane alkaloids in A. belladonna hairy root cultures.