EcoRII Methyltransferase (M.EcoRII) which methylates the second C in the sequence CCWGG (W = A/T) is autogenously regulated by binding to the 5' regulatory region of its gene. DNase I footprinting experiments demonstrated that purified M.EcoRII protected a 47-49 bp region of DNA immediately upstream of the ecoRIIM coding region. We have studied this interaction with mutants of the enzyme, in vitro by DNA binding and in vivo by investigating the repression in trans of expression of beta-galactosidase from an ecoRIIM-lacZ operon fusion. Two catalytically active mutants failed to repress expression of the fusion whereas catalytically inactive mutants had repressor activity. However, with one of the catalytically inactive mutants, C186S, in which the catalytic Cys was replaced with Ser, and which bound unmethylated CCWGG sequences, repression could only be demonstrated when those sequences in cellular DNA were methylated by supplying a cloned dcm gene in trans. In vitro binding of the DNA fragment containing the ecoRIIM regulatory region was detected only with the mutants that showed repressor activity, including C186S. Results indicate that down-regulation of the gene in vivo and binding to the promoter in vitro are not dependent on the catalytic properties of M.EcoRII. Mobility shift experiments with C186S also revealed that it could bind either the promoter or unmethylated CCWGG sites, but not both. We conclude that the concentration of unmethylated CCWGG sites controls expression from the ecoRIIM promoter.