The En/Spm-encoded TNPA protein binds to 12-bp DNA sequence motifs that are present in the subtermini of the transposable element. DNA binding of TNPA to monomeric and dimeric forms of the binding motif was analyzed by gel retardation and cross-linking studies. A DNA binding domain at the N-terminal and a dimerization domain at the C-terminal portion of TNPA were localized using deletion derivatives of TNPA. These domains are novel since no apparent homology has been found in the data bases. The stoichiometry of the TNPA-DNA complexes was analyzed. A special complex is formed with a tail-to-tail dimeric DNA binding motif, most probably involving two DNA-bound TNPA molecules that interact via their dimerization domains. In redox reactions the requirement for one or two disulfide bonds for DNA binding of TNPA was shown. The implications of these findings for the excision mechanism of En/Spm are discussed.