The presence of two distinct host specificities in Staphylococcus aureus strain NCTC8325 was revealed by the isolation of restriction- and modification-deficient mutants. The two host specificity systems, designated S1 and S2, are both active on phage 80mualpha but are not additive in their restricting activity. Restriction-deficient, modification-proficient mutants were invariably affected in both restriction systems. The functional relationship between these two systems is discussed.