Naturally occurring restriction endonucleases are categorized into five groups (Types I, II, III, IV, and V) based on their composition and enzyme cofactor requirements, the nature of their target sequence, and the position of their DNA cleavage site relative to the target sequence.  DNA sequence analysis of restriction enzymes however show great variations, indicating that there are more than four types. All types of enzymes recognize specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific fragments with terminal 5'-phosphates. They differ in their recognition sequence, subunit composition, cleavage position, and cofactor requirements, as summarised below:
* Type I enzymes () cleave at sites remote from a recognition site; require both ATP and S-adenosyl-L-methionine to function; multifunctional protein with both restriction digestion and methylase () activities.
* Type II enzymes () cleave within or at short specific distances from a recognition site; most require magnesium; single function (restriction digestion) enzymes independent of methylase.
* Type III enzymes () cleave at sites a short distance from a recognition site; require ATP (but do not hydrolyse it); S-adenosyl-L-methionine stimulates the reaction but is not required; exist as part of a complex with a modification methylase ().
* Type IV enzymes target modified DNA, e.g. methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA
* Type V enzymes utilize guide RNAs (gRNAs)