The btuB gene of Escherichia coli codes for a protein (BtuB) located in the outer membrane. BtuB is the receptor for vitamin B12 (cyanocobalamin). We have cloned the btuB gene into pUC8 using transposon Tn5 as the marker to first isolate several parts of the relevant DNA fragment from the specialized transducing phage lambda darg13. After reconstitution of the gene, Tn5 was removed by selecting for spontaneous excision. The partial nucleotide sequence and transcriptional start of the btuB gene were determined. The BtuB+ plasmid allowed a large amplification of the synthesis of BtuB, resulting in a 65-fold increased level of vitamin B12 binding. The level of vitamin B12 binding was reduced by a factor of 22 when cells were grown in the presence of high concentrations of vitamin B12. The regulation of the gene was studied in more detail by the use of a protein fusion between the extreme amino-terminus of BtuB and beta-galactosidase of E. coli. The kinetics of repression and derepression were consistent with the presence in the cells of a large amount of a regulatory molecule exhibiting an apparent Km for vitamin B12 of 3 microM.