We have identified important points of contact between the lac repressor and the lac operator by crosslinking the repressor to bromouracil-substituted operator. We substituted bromouracils for thymines in a 55-base-long restriction fragment containing the lac operator and labeled one or the other 5' end with 32P. Ultraviolet irradiation of this fragment produced single-strand breakds at the bromouracils. We examined breakage at each bromouracil in the sequence by denaturing the DNA and displaying the UV-generated fragments on a polyacrylamide gel. In the presence of lac repressor, UV radiation failed to break at specific sites. We attribute this to a competing reaction in which the DNA crosslinks to the repressor rather than breaking. These crosslinkable sites thus define positions at which the lac repressor protein lies close to the methyl group of a thymine in the major groove of DNA.