Fourteen different media were used in the fermentative production of acetone-butanol [by C. acetobutylicum.] The highest total yields were achieved in medium I. Potato starch and soluble starch were suitable as C sources. The best concentrations of potato starch and soluble starch were 500 and 10 g/l, respectively. Peptone was the most favorable N source. The best concentration of peptone was 4 g/l. CaCO3 at 3.6 g/l acted as a buffer in the fermentation process. The best initial pH value of the fermentation medium was 6.0. The optimum temperature was 32.degree.-33.degree. C. The fermentation process required 120 h to obtain maximum yields of acetone-butanol.