Abstract: A number of physicochemical conditions such different concentration of glucose, sucrose, potassium nitrate, ammonium nitrate, calcium chloride and temperatures were tested to optimize growth and production of tropane alkaloids from Datura stramonium (Solanaceae) plants. Cell suspension from semi-clear calli of leave explants developed in MS medium containing kinetin (0.5 mg L-1) and NAA (2 mg L-1) hormones was used to measure biomass and total alkaloids and comparison of treatments. The results showed that 30 and 40 g L-1 glucose led to the highest level of alkaloids and biomass productions, respectively. 20 and 40 g L-1 sucrose concentrations resulted in order the most rates of alkaloids and biomass productions. The results showed that increasing of nitrate concentration led to the reduction of the alkaloids. The best concentration of potassium nitrate for the production of tropane alkaloids and biomass were in order 9.4 and 3.76 mM. Also it was evinced that the optimized concentration of ammonium nitrate for alkaloids production was 10.3 mM and for the biomass was 41.22 mM. The best concentration of calcium chloride for growth and production of the alkaloids was 7.92 mM. Testing different temperature specified that the best condition for production of the alkaloids was 20°C whereas it was 25°C for biomass production. The results of this study could be recommended to farmers involved in production of D. stramonium for tropain alkaloids at industrial and semi-industrial scales.