Introduction Solanum lyratum, a rare species, is used to treat cancer, tumours and warts. Plant cell and tissue culture of S. lyratum, producing steroidal alkaloids, could be useful supplements to natural sources. Objective To study the production of solanine, solanidine and solasodine by adding auxin-type phytohormones including indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) to cell and callus cultures of S. lyratum. Methodology Methanolic extracts were made from callus and cell cultures of S. lyratumand and analysed using RP C18 HPLC with UV detection. Results 2,4-D-induced calli from roots led to a significant enhancement in solanine production with a value of 4.13?mg/g dry weight (DW). The maximal solanidine and solasodine levels of 6.26 and 7.69?mg/g DW were respectively obtained with IBA- and IAA-treated S. lyratum cells at concentrations of 1 and 5?mg/L. Conclusion Auxins were found to be useful phytohormones for the production of steroidal alkaloids. The callus and cell culture system developed is simple and can hence be a method of production of steroidal alkaloids in S. lyratum and other Solanaceae species. Copyright (C) 2011 John Wiley & Sons, Ltd.