Restriction enzymes are generally categorized into four groups, types I, II,III and IV, which differ primarily in structure, cofactor, cleavage site and specificity.

• Type I enzymes: These enzymes cleave at sites remote from a recognition site, which require both ATP and S-adenosyl-L-methionine as cofactors to function. They are multifunctional proteins with both restriction digestion and methylase activities.
• Type II enzymes: They cleave within or at short specific distances from a recognition site. Most of the enzymes fall into this group require magnesium. They are single function protein with only restriction digestion activity.
• Type III enzymes: These enzymes cleave at sites a short distance from a recognition site. ATP is required for this type of enzymes to function. They exist as part of a complex with a modification methylase.
• Type IV enzymes: They recognize and cut modified DNA, typically methylated, hydroxymethylated and glucosyl-hydroxymethylated DNAs.