About 6% of the tested strains of Staphylococcus aureus produced antagonistic substances against staphylococcal indicator strains. The production was low in liquid cultures and could not be induced by ultraviolet irradiation or by treatment with mitomycin C. The antagonistic substances could be classified into at least five groups on the basis of their properties and cross-resistance pattern. One group consisted of lytic enzymes and the four others of staphylococcins. One of the four types of staphylococcins was active against S. aureus only, and the three other types had a broader inhibitory spectrum against gram-positive organisms but not against gram-negative bacteria. A relationship was found between some groups of producing strains and their phage type. The ability to produce staphylococcins was eliminated spontaneously upon storage and more rapidly by treatment with ethidium bromide, acriflavine, acridine orange, sodium dodecyl sulfate, and growth at 42 C. The resistance to several inorganic salts was co-eliminated. No co-elimination of penicillinase production was observed. Selective effects during elimination were ruled out, and the results suggest that the genes for staphylococcin production are plasmid-borne determinants.