The Escherichia coli repressor of biotin biosynthesis, BirA, binds site-specifically to the biotin operator, a 40 base pair imperfect inverted palindrome. Two repressor monomers have been shown to bind to the two operator half-sites. Analysis of results of quantitative DNase I footprint titrations performed on the wild-type biotin operator template indicate that binding is well described by a cooperative mechanism. The data obtained from these studies were, however, insufficient to independently resolve all of the energetic parameters associated with cooperative binding of the two repressor monomers to the operator site. In this work, to further dissect the energetics of assembly of the biotin repressor-biotin operator complex, measurements of binding of BirA to four bioO variants designed to reduce the valency of repressor binding from 2 to 1 have been performed. Results of these measurements indicate, as was found with the wild-type biotin operator template, that two repressor monomers bind simultaneously to the two half-sites of all variant operators. Protein dimerization and DNA binding are thus obligatorily coupled in the biotin repressor system. Furthermore, the results suggest that, in the context of a cooperative binding mechanism, the cooperative free energy associated with the biotin repressor-biotin operator interaction is significantly more favorable than the previously estimated -2 kcal/mol.