Inoculation of leaf sections of Withania coagulans with Agrobacterium tumefaciens strain C58C1 (pRiA4) induced transformed roots with the capacity to produce the most important bioactive compounds of Withania species, withanolide A and withaferin A. The hairy roots obtained showed two morphologies: callus-like roots (CR) with a high capacity to produce withanolides and typical hairy roots (HR) with faster growth capacity and lower withanolide accumulation. The aux1 gene of pRiA4 was detected by PCR analyses in all roots showing callus-like morphology. However, this gene was only detected in 12.5% of the roots showing typical hairy root morphology. This fact suggests a significant role of aux genes in the morphology of transformed roots. Time course studies of withanolide production showed that withanolide A accumulated during the first part of the culture whereas the maximum accumulation of withaferin A occurred at the end of the culture period. Some transformed root lines, such as HR112 and CR26, showed considerable potential to produce withanolides in a scaled up bioreactor system, especially the important pharmaceutical compound withanolide A.