We measured quantitatively the binding affinities of purified Cro repressor to the chemically synthesized wild-type and mutant OR1 operators, consisting of all three possible base-pair substitutions and of thymine to uracil substitutions at each base-pair position of the 17-base-pair operator sequence. The sequence-specific interactions between Cro repressor and the operator DNA occur at the symmetrically disposed outer 7-base-pair positions of each half operator and at the central base-pair position. The binding of Cro is almost symmetrical with respect to the pseudo-twofold symmetry of the binding site. The binding free energy changes calculated from the affinity changes are mostly additive for specific Cro binding. Also the binding affinities of Cro to the operators or any other DNA sequences can be predicted by simple addition of free energy changes of single base substitutions. We isolated cro mutants by site-directed mutagenesis and studied their DNA binding to the wild-type and base-substituted mutant operators. The sequence-specific contacts derived from such studies are significantly different from the models proposed by Ohlendorf et al. [Ohlendorf, D. H., Anderson, W. F., Takeda, Y. & Matthews, B. W. (1982) Nature (London) 298, 719-723] and by Hochschild et al. [Hochschild, A., Douhan, J., III, & Ptashne, M. (1986) Cell 47, 807-816].