Rifamycin B production by isolated Amycolatopsis sp. RSP 3 was investigated under solid state fermentation (SSF) using agro-industrial waste materials. Corn husk was the Most Suitable substrate/support material with 4-fold higher production than wheat bran and corn cobs. A two-level (conventional and statistical) methodology was used to optimize fermentation parameters belong to physiological (pH, temperature and aeration), nutritional (carbon and nitrogen sources) and microbial (inoculum level and incubation time). Conventional optimization significantly improved (450%) the rifamycin B production of which two-third was associated with carbon and nitrogen sources. Starch as carbon source showed negative impact. Statistical optimization of suggested potassium nitrate (at individual level), soya bean meal and barbital (at interactive level) were observed to be the most noticeable variables in the maximization of production. At optimized conditions, inorganic nitrogen source played vital role (>59%) compared to all other factors. Overall, more than 920% increase in rifamycin B production was achieved at optimized environment. (C) 2009 Published by Elsevier Ltd.