The Escherichia coli chromosome encodes two methylases, dam and dcm, which recognize the sequences GATC and CC(A/T)GG, respectively. Specific dam and dcm sites on the E. coli chromosome were found to be unmethylated in vivo by using pulsed-filed gel electrophoresis experiments scanning megabase regions of DNA. Some sites were totally unmethylated. The dam sites display variable methylation depending on the local sequence, and, in general, their methylation shows complex modulation by growth conditions and growth rate, suggesting multiple protection mechanisms. Sites resistant to complete dam or dcm methylation appear to be distributed throughout the chromosome. These unusual sites may identify regions of the chromosome with interesting biological functions.