The Pvu II restriction endonuclease (R. Pvu II) cleaves CAG downward arrowCTG sequences as indicated, leaving blunt ends. Its cognate methyltransferase (M. Pvu II) generates N4-methylcytosine, yielding CAGN4mCTG, though the mechanism by which this prevents cleavage by R. Pvu II is unknown. The heterologous 5-methylcytosinemethylation CAG5mCTG has also been reported to prevent cleavage by R. Pvu II and this has been used in some cloning methods. Since this heterologousmethylation occurs at the native methylated base, it can provide insights into the detection of DNAmethylation by R. Pvu II. We found that the cloned gene for R. Pvu II could not stably transform cells protected only by M. Alu I (AG5mCT) and then determined that R. Pvu II cleaves CAG5mCTG in vitro, even when both strands are methylated. DNase I footprint analysis and competition experiments reveal that R. Pvu II binds to CAG5mCTG specifically, though with reduced affinity relative to the unmethylated sequence. These results provide biochemical support for the publishedstructures of R. Pvu II complexed with DNA containing CAGCTG and CAG5-iodoCTG and support a model for how methylation interferes with DNA cleavage by this enzyme.