A method based on the differential rate of cytosine methylation in single- and double-stranded nucleic acids by dimethyl sulfate [Peattie, D.A. & Gilbert, W. (1980) Proc. Natl. Acad. Sci. USA 77, 4679-4682] has been developed for probing unpaired cytosines in DNA and DNA-protein complexes at the sequence level. Application of the method to the complexes between Escherichia coli RNA polymerase (EC 2.7.7.6) and three related promoters, lac UV5, trp, and a hybrid promoter tac resulting from the fusion of the two, reveals distinct differences in the way RNA polymerase unpairs DNA in these promoters. No single-stranded region is detectable in the complex with the trp promoter. For the lac UV5 promoter, the cytosines at positions -6, -4, -2, and -1 are in an unpaired region. The same cytosines in the tac promoter, which is homologous in sequence to lac UV5 in this region, are also found to be single stranded. For the pair of promoters lac UV5 and tac, the cytosine methylation reaction has also been used to demonstrate the steep temperature dependence of opening of base pairs by RNA polymerase. One striking feature is that the midpoint of this transition for the tac promoter is 3 degrees C lower than the corresponding value for lac UV5, even though the sequence of the unpaired region in the two promoters is identical.