The aim of this study was to optimize the culture conditions for keratinase production by a new isolate, Streptomyces sp. 2M21 utilizing chicken feather as an alternative low-cost substrate. Contribution of different independent fermentation variables on keratinase activity was investigated by Plackett-Burman design. Parameters that have the highest contribution were selected for the optimization experiments which were carried out by using response surface methodology (RSM). Relative and mutual effects of fermentation variables on keratinase activity were analyzed with "2(3) Full Factorial Central Composite Design". Cultivation processes were implemented in 250 ml Erlenmeyer flasks containing 50 ml medium. Maximum keratinase activity was obtained under optimal conditions of 28 degrees C, 5.0 g l(-1) chicken feather and 5.5 days of process time. As a result of the optimization studies, a 15 fold increase in keratinase activity was reached compared to the unoptimized conditions. Furthermore, similar results were obtained in the 2 1 bioreactor under optimized conditions. (c) 2015 Elsevier Ltd. All rights reserved.