The chromosomal location of two genetic loci involved in the transport of cyanocobalamin (B(12)) in Escherichia coli K-12 was determined. One gene, btuA, is believed to code for the transport protein in the cytoplasmic membrane, because a mutant with an alteration in this gene has lost the ability to accumulate B(12) within the cell although normal levels of the surface receptors for B(12) are present. The other locus, btuB, apparently codes for the surface receptor on the outer membrane. These mutants have lost the ability to bind B(12) and have greatly reduced transport activity, although growth experiments have shown that they can utilize B(12) for growth, but with decreased efficiency. This surface receptor for B(12) also appears to function as the receptor for the E colicins, because btuB mutants are resistant to the E colicins, and mutants selected for resistance to colicin E1 are defective in B(12) binding and transport. The gene order was determined by transduction analysis to be cyc-argH-btuA-btuB-rif-purD. In addition, mutations in metH, the gene for the B(12)-dependent homocysteine methylating enzyme, were obtained in this study. This gene was localized between metA and malB.