Eight strains producing bacterial cellulose (BC) were isolated from rotten fruits and traditionally fermented vinegars. One of the isolated strains from the rotten grape in Gwangju, Korea, maintained a relatively stable BC production in shaking cultures. This isolated strain proved to be Acetobacter xylinum, based on several biochemical and morphological tests. It was shown that the slant-baffled flask was more efficient than the conventional flask for the BC production in shaking cultures. To determine the most suitable carbon and nitrogen sources for the production of BC, various compounds were examined. Fructose was found to be the most effective carbon source with an optimal concentration of 2%. Mixed carbon source (glucose:fructose=1:3) was also better than glucose or fructose alone. Optimal nitrogen source, when basal medium was used, was 10% (v/v) com steep liquor (CSL). When com steep liquor was used with a mixed carbon source (glucose:fructose=1 :3),4% CSL exhibited the best BC production. Based on these results, a defined medium was developed for the BC production by Acetobacter xylinum KJ-1. When this medium was used under optimal culture conditions, the BC production was 7.2 g/1, which was approximately 3 times higher than that with the traditional HS medium.