Fok I restriction endonuclease recognizes the nonpalindromic pentadeoxyribonucleotide 5'-GGATG-3'.5'-CATCC-3' in duplex DNA and cleaves 9 and 13 nt away from the recognition site. Recently, we reported the presence of two distinct and separable domains within this enzyme: one for the sequence-specific recognition of DNA (the DNA-binding domain) and the other for the endonuclease activity (the cleavage domain). Here, we report the construction of a chimeric restriction endonuclease by linking the Drosophila Ultrabithorax homeodomain to the cleavage domain (FN) of Fok I restriction endonuclease. The hybrid enzyme, Ubx-FN, was purified, and its cleavage properties were characterized. The hybrid enzyme shows the same DNA sequence-binding preference as that of Ubx; as expected, it cleaves the DNA away from the recognition site. On the 5'-TTAATGGTT-3' strand the hybrid enzyme cleaves 3 nt away from the recognition site, whereas it cuts the complementary 5'-AACCATTAA-3' strand 8, 9, or 10 nt away from the binding site. Similarly engineered hybrid enzymes could be valuable tools in physical mapping and sequencing of large eukaryotic genomes.