TnpA protein, the function encoded by the most abundant transcript of En-1 was expressed in Escherichia coli. DNA binding experiments with partially purified tnpA protein revealed that it binds to the subterminal repetitive region of En-1. TnpA protein recognizes a 12-bp-long sequence motif which is reiterated several times at the termini of En-1. Binding is reduced if the cytosine residues of CG dinucleotides and CNG trinucleotides within the motif are methylated. These data suggest a model in which the product of tnpA serves as a regulator of element activity.