Hairy root cultures of Datura quercifolia were established following infection with Agrobacterium rhizogenes strain LBA 9402. Eight tropane alkaloids were identified in the hairy roots, hyoscyamine being the major comtituent. The growth and the hyoscyamine content of transformed roots were investigated under various conditions. Gamborg B5, Mun,.shige and Skoog, and Woody Plant media were tested. Gamborg B5 medium was the best for growth as well as for hyoscyamine accumulation. The influence of sm;rose concentration was examined and a 5 % concentration was found to be the most appropriate for growth and for alkaloid production. After 35 days of incubation in this medium, the hyoscyamine content of the roots was 1. 24 % based on dry weight. The influence of gibberellic acid and of Amberlite XAD-4 resin on hyoscyamine production was tested.