The occurrence of class II restriction endonucleases (enzymes that both recognize and cleave a specific nucleotide sequence in deoxyribonucleic acid [DNA] in Staphylococcus aureus has been investigated by analysis of crude extracts obtained from different propagating strains of the International Phage Typing System. Of the four main groups of strains in the International System, only extracts of group II strains were found to contain class II restriction endonucleases. The identical cleavage patterns obtained by incubation of different DNAs with cell extracts of group II strains suggest that these enzymes all recognize and cleave the same nucleotide sequence. This recognition site has been determined to be 5'-G-A-T-C-3'-3'-C-T-A-G-5' for the prototype of these enzymes, Sau3AI (J. S. Sussenbach et al., Nucleic Acids Res. 3:3192-3202, 1976). Evidence is presented that the classification of group II strains is based on restriction modification and is correlated with the presence of a class II restriction enoduclease that recognizes and cleaves the above sequence.