Fractionation on Sephadex G-25 column of free and bound vit. B12, added to or contained in the normal human gastric juice, has achieved a good separation of free and bound B12- C060B12 or Co57B12 was added to the solutions of dialyzed and lyophilized gastric juice in borate buffer (pH 9.0, 0.06-0.24) (50-113 mug labelled B12 per mg of gastric solids) and eluted from the columns with the same buffer. The total bed volumes of the columns ranged from 32 to 114 ml, and thier Vo - from 11 to 43 ml. Vitamin B12 eluted into the second peak of the effluent represents free B12 only. This is shown by elution pattern of free B12 into this fraction only, complete dialyzability of B12 contained in this fraction; complete adsorption of B12 from this fraction by charcoal and characteristic cathodic electrophoretic mobility of B12 contained in this fraction. Conversely, B12 eluted into the first peak represents bound B12 only, since radioactivity is here associated with the presence of tyrosine con-taining materials, B12 is here non-dialyzable, and it shows anodic mobility only on paper electrophoresis in borate buffer, characteristic for B12 bound to B12-binders in human gastric juice. Excellent yield of materials eluted was achieved and many other advantages of this method of fractionation of free and bound B12 became apparent.