We have used localized mutagenesis of the biotin domain of the Escherichia coli biotin carboxyl carrier protein coupled with a genetic selection to identify regions of the domain having a role in interactions with the modifying enzyme, biotin protein ligase. We purified several singly substituted mutant biotin domains that showed reduced biotinylation in vivo and characterized these proteins in vitro. This approach has allowed us to distinguish putative biotin protein ligase interaction mutations from structurally defective proteins. Two mutant proteins with glutamate to lysine substitutions (at residues 119 or 147) behaved as authentic ligase interaction mutants. The E119K protein was virtually inactive as a substrate for biotin protein ligase, whereas the E147K protein could be biotinylated, albeit poorly. Neither substitution affected the overall structure of the domain, assayed by disulfide dimer formation and trypsin resistance. Substitutions of the highly conserved glycine residues at positions 133 and 143 or at a key hydrophobic core residue, Val-146, gave structurally unstable proteins.