Atropa belladonna L is most important commercial source of pharmaceutical tropane alkaloids. Initiation of callus culture on MS solid media with different concentrations of growth regulators as BA and 2,4 D from different explants shows that 2,4-D only at 1.0mg/L and 2.0mg/l gave the highest callus formation score after 21 days. The highest concentration of atropine (376.62 µg/g DW) and scopolamine (103.16 µg/g DW) were obtained from leaf callus on MS medium supplemented with 2,4 D at 0.5 mg/L after 28 days. The effect of elicitors and precursor feeding on tropane alkaloids production in callus culture were examined. Accumulation of both alkaloids; atropine and scopolamine in callus were enhanced after 8 days with jasmonic acid at concentrations (50 µM), after 15 days with yeast extract at concentrations (0.5 g/L) and after 21days with ornithine at 1mM in comparison with control callus. Salicylic acid inhibits callus growth and accumulation of atropine and scopolamine in treated callus.