Publisher Summary This chapter discusses the recognition of DNA by type II restriction enzymes. A restriction/modification (R/M) system must possess two enzyme activities, the restriction endonuclease and the modification methylase, both of which are dependent on the recognition of the same DNA sequence. In each R/M system, the modification activity results in the methylation of a particular base within a specific DNA sequence, usually the conversion of an adenosine to N 6 -methyladenosine or a cytosine to 5-methylcytosine depending on the R/M system in question. The restriction activity cleaves the DNA but only if the recognition sequence is not methylated. Hence, the bacterium can discriminate between DNA that is not methylated at the recognition sequences and DNA that is methylated at these sites. Many different R/M systems have now been characterized, initially by genetic analyses and subsequently by the purification of the proteins. Both criteria revealed the existence of three distinct classes of R/M systems, and these are now categorized as types I, II, and III. Type II restriction enzymes are endonucleases that recognize specific DNA sequences and in the presence of Mg 2+ , they cleave both strands of the DNA at fixed locations relative to the recognition sequence. At present, over 640 different Type II restriction enzymes have been identified from a wide variety of bacterial genera and between them, they recognize over 135 different DNA sequences. Furthermore, the discrepancy between the numbers of restriction enzymes and recognition sequences arises from the observation that restriction enzymes from different bacteria are frequently found to recognize the same DNA sequence.