Abstract A hairy root culture of Duboisia myoporoides was established by transforming cultured shoots with Agrobacterium rhizogenes strain HRI. Approximately one month after inoculation with HRI, a shoot produced many roots at the inoculation point. The roots, designated AR-4, were transferred to Linsmaier-Skoog liquid medium without growth regulators and were sub-cultured every three weeks. Opines, agropine and mannopine were detected in the extract of AR-4 cells. The scopolamine content of AR-4 was lower than that of untransformed roots (RB-1), whereas the hyoscyamine content was higher. Cell division took place during the culture of AR-4; the cell yield of AR-4 was 2-fold that of RB-1 after three weeks of culture. The concentration of endogenous indoleacetic acid in the AR-4 cells was not very high as compared with that of the RB-1 cells. The addition of indoleacetic acid or indolebutyric acid to the medium had little effect on the production of tropane alkaloids by AR-4, whereas 2,4-dichlorophenoxyacetic acid and benzyladenine decreased alkaloid production.