Genetic experiments have suggested that the lexA gene product of Escherichia coli represses a number of genes involved in the response to DNA damage, including recA and lexA. We purified the lexA gene product from bacterial strains that bear plasmids that direct the synthesis of large amounts of the protein. Purified lexA protein bound to two symmetrical DNA sequences in front of lexA and one in front of recA, protecting them from digestion with DNase I and blocking methylation of purines in the major groove. lexA protein repressed transcription of both genes in vitro. lexA protein binds to the two sites in front of the lexA gene with approximately the same affinity and with greater affinity to the single site in front of the recA gene. The affinity of lexA protein for its operator sites was measured under conditions that mimic conditions in vivo. Differences in the affinity with which lexA protein binds to the operators of genes it represses may account for the differences in the timing and extent of their induction after DNA damage.