A biotin‐containing hexapeptide Ac‐Glu‐Ala‐Met‐Bct‐Met‐Met (1) that represents the local biotin‐containing site ofEscherichia coliacetyl‐CoA carboxylase has been prepared by the solid phase method. Peptide1is carboxylated by the biotin carboxylase subunit dimer ofE. coliacetyl‐CoA carboxylase with the following kinetic parameters; Km12 mm, Vmax2.8 μm· min–1. These compare with the parameters for biotin of Km214 mmand Vmax28 μm· min–1. Hence, the overall reactivity (Vmax/Km) of1is 1.8 times greater than that of free biotin. When all methionines in1are replaced by alanine, the resulting peptide (2) retains a similar binding ability but with a much decreased Vmax. It was also found that peptide3, which carries anN‐benzyloxy carbonyllysine in place of biocytin in1, decrea