Acetyl CoA carboxylase of Escherichia coli has been resolved into three functionally dissimilar proteins: ( 1 ) biotin-carboxyl carrier protein (BCCP); ( 2 ) a biotin carboxylase component that catalyzes the Mn-ATP-dependent carboxylation of BCCP to form CO 2 - -BCCP; and ( 3 ) a transcarboxylase component that catalyzes the transfer of the carboxyl group from CO 2 - -BCCP to acetyl CoA to form malonyl CoA. The transcarboxylase has been purified 1700-fold. Evidence that this protein catalyzes the transcarboxylase step includes the demonstration that it ( a ) catalyzes the carboxylation of BCCP, ( b ) catalyzes the BCCP-dependent exchange between [ 14 C]acetyl CoA and malonyl CoA, ( c ) binds labeled acetyl CoA and malonyl CoA, and ( d ) catalyzes the decarboxylation of CO 2 - -BCCP. On the basis of this evidence, it is concluded that the transcarboxylase component contains sites for the acyl CoA group and for biotin, the covalently bound prosthetic group of BCCP.