The chemical and biological properties of vitamin B 12 combining substances in mouse stomach extracts (MIF), mouse serum (MS), and mouse ascites fluid (AS) were compared. MIF was found to enhance the absorption of vitamin B 12 by rings of mouse and guinea pig ileum and to inhibit its absorption by sarcoma-180 (S-180) cells in vitro. MS and AS, on the other hand, enhanced the absorption of vitamin B 12 by S-180 cells but inhibited its absorption by rings of mouse and guinea pig ileum. It was shown that MIF is capable of forming a stable complex with vitamin B 12 at pH values from 1.0 to 11.0, while MS and AS are not capable of forming such stable complexes at pH values below 5.0. The complex formed between vitamin B 12 and MIF (MIF-vitamin B 12) is not adsorbed by zirconyl phosphate gel (Z-gel) at pH values higher than 5.0; however, the complexes formed between vitamin B 12 and MS (MS-vitamin B 12) or AS (AS-vitamin B 12) are adsorbed by Z-gel at pH values as high as 7.0. By chromatography on CM-cellulose, it was possible to separate MIF-vitamin B 12 from MS-vitamin B 12 and AS-vitamin B 12. The latter two complexes behaved identically when chromatographed on CM-cellulose.