The establishment of de novo patterns of DNA methylation in mammalian genomes is characterized by the gradual spreading of methylation, which has been documented to occur across an entire integrated adenovirus genome as well as at the nucleotide level in the integrated late E2A promoter of adenovirus type 2. By applying the techniques of genomic sequencing and dimethylsulfate or DNase I genomic footprinting in vivo, we have now demonstrated that the spreading of methylation in cell lines that carry the late E2A promoter with three in vitro pre-methylated 5'-CCGG-3' sequences initially involves a DNA domain of this promoter that is devoid of bound proteins. Subsequently, methylation further spreads to neighboring regions, and the patterns of complexed transcription factors are altered. Evidence has been adduced that DNA methylation at sequences homologous to the AP-1 and octamer binding factor sites interferes with protein binding. In contrast, the methylation of sequences in the vicinity of but not involving sequences homologous to an AP-2 site still permits the binding of proteins to these sites. It is significant that during the spreading of methylation a few 5'-CG-3' sequences can remain hemimethylated for several cell generations, before they also become methylated in both complements. Moreover, in cell line HE2, the integrated, heavily methylated late E2A promoter has been shown by the genomic sequencing technique to contain 5-methyldeoxycytidine residues, not only in all 5'-CG-3' dinucleotides but also in a 5'CA-3' and a 5'-CT-3' dinucleotide sequence. Hence, 5-methyldeoxycytidine occurs in a silenced mammalian DNA sequence also in dinucleotides other than 5'-CG-3'. This finding raises the question of whether 5-methyldeoxycytidine in non-5'-CG-3' dinucleotides can be maintained in the methylated state during continuous cell propagation.