The native forms of vitamin B12-active substances and the natural occurrence of Barker''s cobamide coenzyme in the cells of methane bacteria are not entirely known. Most of non-cyano type vitamin B12 were transformed to the cyano type by the extraction procedure using cyanide solution. An extraction method with hot ethanol was used instead of the cyanide treatment to avoid the transformation of cyanide-labile vitamin B12 analogues. A purified extract was obtained by the usual phenol treatment of the alcohol extract. By paper ionophresis performed in 0. 5 N acetic acid, 5 zones active to E. coli No 215 were observed: 3 of them (tentatively named P1-P3 in the order of the mobilities) moved toward the cathode, one (P4) stayed near the start and the fifth (P5) migrated toward the anode. Disappearance of P1 and P2 and increase in the area of P4 were observed in the extract with cyanide. In the bioautogram using Ochromonas malhamensis which has more specific response to 5, 6-dimethylbenzimidazolyl cobamide than E. coli, the growth zones corresponding to P2 and P3 were not detected. The partially purified substance was fractionated by DEAE-cellulose column chromatography. A fraction eluted with water contained the vitamin B12-active substance corresponding to the zone P1. The fraction exhibited the coenzyme activity in the propanediol-propionaldehyde conversion. The P1 corresponds to 5, 6-dimethylbenzimidazolyl cobamide coenzyme (DBCC) P2, non-cyano type B12 analogue lacking 5, 6-dimethylbenzimidazolyl cobamide structure; P3, pseudocobalamin; P4, cyanocobalamin and P5, an unidentified B12 analogue with anionic character.