In the regulation of eukaryotic gene expression, the interactions of several protein factors with specific signals in the promoter sequence can play a decisive role. In addition, the methylation of specific promoter sequences causes the long-term inactivation of eukaryotic promoters. The function of the regulatory signal 5-methylcytidine is complex and can be overcome by a number of factors, e.g., by the E1 proteins of adenoviruses. We studied the inactivation of the late E2A promoter of adenovirus type 2 (Ad2) DNA by the methylation of three 5'-CCGG-3' sequences at positions -215, +5, and +23, relative to one of the cap sites of this promoter. One would like to understand the mechanisms by which 5-methylcytidine residues are capable of interfering with regulatory functions in the late E2A promoter of Ad2. We have identified six different promoter sequences by DNase I protection analyses, and have shown that these sites bind specifically to host proteins (binding sites I-VI). Binding of these factors to unmethylated or 5'-CCGG-3' methylated late E2A promoter sequences was compared by gel migration delay assay or DNase I protection (footprinting) analyses. Protein binding does not appear to be affected by late E2A promoter methylation. Even after partial purification of some of these factors by chromatography on heparin-Sepharose, differences in binding to the unmethylated and the 5'-CCGG-3' methylated promoter were not observed. Even though striking differences in host factor binding were not detectable at late E2A promoter sites, it is conceivable that the functionality of promoter-bound host proteins is altered when the three 5'-CCGG-3' sequences are methylated.