Abstract The uptake of cyanocobalamin (CN-B12) by cells of Escherichia coli consists of an initial rapid phase of B12 binding to specific receptors on the outer membrane of the cell envelope, which is followed by a slower secondary phase in which the B12 is transferred from these receptors into the interior of the cell. This paper describes some of the properties of the initial phase of uptake in cells of E. coli KBT001, a methionine-B12 auxotroph, and in cells of B12 transport mutants derived from this strain. Cells of strains KBT026 and KBT069 lacked the initial phase of B12 uptake, whereas those of strains KBT041 and KBT103 were defective only in some part of the secondary phase. The kinetic characteristics of the initial phase of uptake were indistinguishable in cells from strains KBT001, KBT041, and KBT103, giving a Km for CN-B12 of about 4 nm. Competitive inhibition of this uptake was given by aquocobalamin, cyanocobinamide, methyl cobalamin, and deoxyadenosyl cobalamin, with Ki values ranging from 0.3 to 4.2 nm. No inhibition was given by cyanocobalamin 5'-phosphate, which was the only corrinoid compound tested which was apparently not recognized by the initial B12-binding sites. Cells from strains KBT001, KBT041, and KBT103 each possessed approximately 200 initial B12-binding sites per cell. These sites were found to be firmly bound to the outer membrane of the cell envelope and were not released by osmotic shock treatment of whole cells, sonication, sucrose density gradient centrifugation of envelope fragments, selective solubilization of the inner membrane by treatment of whole envelopes with Triton X-100-MgCl2, and exposure of cell envelopes to chaotropic agents or enzymes such as lysozyme, trypsin, and phospholipases A, C, and D. Cell envelopes and outer membrane preparations from cells of strains KBT026 and KBT069 had no detectable ability to bind CN-[60Co]B12. The B12-binding activity in the outer membrane was solubilized by extraction with 2% Triton X-100-1 mm EDTA at pH 7.6 with retention of about 50% of its activity. The binding of CN-B12 to whole cells, cell envelopes, outer membrane particles, and solubilized outer membranes was inhibited by 1 mm EDTA and 1 mm ethylene glycol bis(β-aminoethyl ether)-N,N'-tetraacetic acid, but not by 5 mm o-phenanthroline. This inhibition was reversed by a variety of divalent cations, the most effective of which was Ca2+. Osmotic shock treatment of the cells released trace amounts (5 to 10 binding sites per cell) of B12-binding activity, which was separated into two distinct size fractions by chromatography on Sephadex G-100. The larger of these species was found only in cells from strains which possessed an intact initial phase of B12 uptake and is believed to consist of small fragments of the outer membrane containing l5% of the initial B12-binding sites. The small B12-binding species was detectable in all of the strains, but no clear role for it in B12 transport has yet been established. The inner membrane from the E. coli cell envelope was unable to bind free CN-B12, and it was concluded that the presumptive B12 receptor on this membrane must recognize B12 only when the B12 is attached to some specific donor molecule. This would indicate an essential similarity between the B12 uptake systems of the E. coli inner membrane and mammalian plasma membranes. Possible candidates for such a B12 donor are the outer membrane B12 receptor and the smaller B12 binder in the periplasmic space.