Publisher Summary This chapter discusses the preparation and properties of immobilized sequence specific endonucleases. The type II restriction endonucleases have become valuable reagents for research in molecular biology. This is primarily due to their unique property of cleaving DNAs at a limited number of specific sites. These enzymes have been employed for use in physical DNA mapping, plasmid construction, and gene cloning. Most of these enzymes recognize and cleave DNA at specific sequences, which are usually in the form of a palindrome. There is evidence implying that these endonucleases are membrane bound, therefore, one approach to study them is to determine what effect in-solubilization (that is, mimicking membrane binding) has on their activity. The immobilized enzymes can be packed in the form of a column or rapidly pelleted from solutions by centrifugation. These procedures allow quick removal of restriction endonucleases from reaction mixtures. Such recovered enzymes are reusable several times over, permitting cleavage of large amounts of DNA with relatively few units of enzyme. Furthermore, the chapter describes the catalytic and functional characterization of covalently bound restriction endonucleases.