The relative binding affinities of Mnt protein from bacteriophage P22 are determined for each possible base pair at position 17 of the operator. These are determined from the partitioning of randomized operators into bound and unbound fractions; quantitation is provided by restriction enzyme analysis. Mnt protein is found to have an unusual specificity at this position: a C.G base pair (the wild-type operator) has the highest affinity, a G.C base pair has the lowest affinity, and both orientations of A.T base pairs are intermediate and nearly equivalent. A specific binding constant and specific binding free energy are defined and shown to be directly related to the information content of the operator sequences bound to the protein, taking into account the quantitative differences in binding affinities.