Azadirachtin, a well-known biopesticide, is a secondary metabolite extracted from the seeds of Azadirachta indica. In the present study, azadirachtin was produced in hairy roots of A. indica, generated by Agrobacterium rhizogenes-mediated transformation of leaf explants. Liquid cultures of A. indica hairy roots were developed with a liquid-to-flask volume ratio of 0.15. The kinetics of growth and azadirachtin production were established in a basal plant growth medium containing MS medium major and minor salts, Gamborg's medium vitamins, and 30 g l -1 sucrose. The highest azadirachtin accumulation in the hairy roots (up to 3.3 mg g -1) and azadirachtin production (~44 mg l -1) was obtained on Day 25 of the growth cycle, with a biomass production of 13.3 g l -1 dry weight. To enhance the production of azadirachtin, a Plackett-Burman experimental design protocol was used to identify key medium nutrients and concentrations to support high root biomass production and azadirachtin accumulation in hairy roots. The optimal nutrients and concentrations were as follows: 40 g l -1 sucrose, 0.19 g l -1 potassium dihydrogen phosphate, 3.1 g l -1 potassium nitrate, and 0.41 g l -1 magnesium sulfate. Concentrations were determined by a central composite design protocol and verified in shake-flask cultivation. The optimized medium composition yielded a root biomass production of 14.2 g l -1 and azadirachtin accumulation of 5.2 mg g -1, which was equivalent to an overall azadirachtin production of 73.84 mg l -1, 68% more than that obtained under non-optimized conditions. © 2011 The Society for In Vitro Biology.