The positions of interference points between the IclR repressor of the acetate operon of Escherichia coli and its specific operator were examined. The number and nature of nucleotides essential to repressor binding were determined by scanning populations of DNA previously methylated at guanine residues by dimethyl sulfate, or depurinated by treatment with formic acid, or depyrimidated by treatment with hydrazine. A total of 46 nucleotides, distributed almost equally between the two strands of the operator region, were found to be functionally important, although to a varying extent. These are clustered in two successive domains which expand from nucleotide -54 to nucleotide -27 and can organize in a palindrome-like structure containing a large proportion of A and T residues.