The effects of tween 20 as permeabilizing agent on tropane alkaloids from Datura innoxia Mill, hairy roots have been studied. For various tween 20 concentrations both hyoscyamine and scopolamine accumulated in the culture medium. Plant material viability could be preserved after a 24 hours-2% tween 20 concentration treatment. The time-course study of alkaloid release showed that the maximum of excretion occurred after a 20 hour contact with tween 20. At that time, a concentration of hyoscyamine superior to 25 mg/l was detected in the medium.