Two extremely oxygen-sensitive strains of Clostridium sp., designated Clostridium E and P, were obtained from digestive microflora of conventional mice and found to constitute a barrier against Shigella flexneri SF-2 when associated in vivo with Escherichia coli K-12. These and other simplified fractions of the conventional microflora were demonstrated to have an effect comparable to that of the total flora. When K-12 and Clostridium E were established in gnotobiotic mice before the introduction of SF-2, the latter was reduced to a level below detection in the digestive tract. Whe SF-2 was established first, the antagonistic effect exerted by Clostridium E and K-12 was variable and, apparently, related to the rate of establishment of Clostridium E. Mutants of SF-2 resistant to the barrier effect of Clostridium E and K-12 appeared at the end of 3 months when SF-2 was established in gnotobiotic mice alone or with K-12, and after only a week when SF-2 was associated only with Clostridium E. These results suggest that the bacterial antagonsim in this model is related to the production in vivo of an antibiotic substance active against SF-2. It appears that the substance may be produced by Clostridium E, stimulated by K-12.