For the purpose of finding out a strain which has stronger cytolytic activity on Ehrlich ascites carcinoma cells, the authors isolated 113 strains of Bacillus natto from straws collected at various areas in Japan and measured the cytolytic activities by cylinder plate method. As the results, a strain tentatively called KMD 2311 which has the strongest cytolytic activity in the 113 strains was found out. There were at least two kinds of cytolytic substances on Ehrlich ascites carcinoma cells in the culture medium of Bacillus natto KMD 2311. One was extracted with AcOEt from the culture medium, which constitute approximately 20% of the cytolytic activity in the culture medium and it was stable. This cytolytic substance was purified and a colorless crystalline compound (mp 247-249°) was obtained by recrystallization from acetone-petr. ether. Chemical structure of this compound was examined by elementary analysis, infrared, nuclear magnetic resonance, and mass spectroscopy and study of decomposed products. As the results, this compound was proved to be identical with surfactin (mp 140°), which was obtained from culture medium of Bacillus subtilis by Kakinuma, et al. and from Bacillus natto KMD 1126 by the authors. Melting point of this compound (mp 249°) was higher about 100° than that of surfactin (mp 140°), but high mp compound was obtained from surfactin by recrystallization from acetone-petr. ether. From these results, it was indicated that the cytolytic substance was identified with surfactin and dimorphic.