Cell suspension cultures of Withania somnifera were established in shake flasks and the effect of different growth regulators (auxins, combination of auxin and cytokinin), inoculum density (2.5-20 g L(-1)), different media (MS, B5, NN and N6), the strength of the MS medium (0.25-2.0x), carbon source (sucrose, glucose, fructose, maltose), concentration of the sucrose (1-8% (w/v)) and the initial pH (4.0-6.5) of the medium were determined for the production of withanolide A. The optimized conditions for biomass accumulation and withanolide A production were found to be 10 g L(-1) of the inoculum on fresh weight basis, the full strength MS medium, 3% (w/v) sucrose, four weeks culture period and the initial medium pH of 5.8. The results of present study are useful for scale-up process. (C) 2010 Elsevier Ltd. All rights reserved.