The study was aimed to optimize the culture conditions for the production of usnic acid in the cultured cell aggregates composed of symbionts in lichen Usnea ghattensis in vitro. The cultured lichen tissue composed of symbionts appeared after about 2-3 weeks of inoculation in water-agar and malt-yeast extract (MYE) media and shown the production of usnic acid after 2-3 months of inoculation. However, the growth of symbionts was strongly affected by different culture conditions. The addition of excess carbon and nitrogen sources in the media has significantly enhanced the growth as well as usnic acid content. The cultured symbionts in MYE medium having 4% sucrose, 4% polyethyl glycol (PEG) gave 7.63 g dry biomass with 3.9 microg usnic acid/g dry biomass. In water-agar medium having 4% sucrose and 4% PEG gave 3.08 g dry biomass with 1.11 microg usnic acid/g dry biomass. The positive effects of medium on the growth of symbionts and the production of usnic acid are seemed to be due to nutritional factors.