Solanaceous plant, Datura innoxia, has valuable tropane alkaloids pharmaceutical effects. In this study, seed zygotic embryos were cultured in vitro and plantlets were produced and explants with single internodes and two auxiliary buds from the produced plantlets were cultured in B5 medium with different concentrations of IAA (0.5, 1, 1.5, 2 mg/L), NAA (0.5, 1, 1.5, 2 mg/L) and BA (0.5, 1, 1.5 mg/L) to investigate the effects of the growth regulators on callus production, organogenesis and total alkaloid contents. Concentrations of the last two hormones were used reciprocally. Total alkaloid concentrations of calli were identified by UV spectrophotometer at 258 nm and callus total alkaloid contents and were compared with the content of plantlets. Results showed that, in the presence of both NAA and BA, explants formed callus and increase of NAA concentrations, in turn, increased callus weights and total alkaloid contents as well. IAA had no significant effect on callus and organ induction. Comparison of total alkaloid contents of calli with shoots and roots of Datura innoxia plantlets in hormone free B5 medium, showed that the alkaloid contents of calli were lower than differentiated tissues in hormone free B5 medium, but the use of NAA and BA hormones caused an increase in alkaloid contents of calli as compared with in vitro cultured organs.