Screening of non-phage group II Staphylococcus aureus strains for antagonistic substances revealed one particular strain, S. aureus D91, to excrete a substance with a wide spectrum of activity; both Gram-positive and Gram-negative bacteria were susceptible. The staphylococcin-like substance D91 produced by this strain was partially purified by column chromatography on Sephadex G-50, DEAE-cellulose, Phenyl Sepharose CL-4B and Sephadex G-200. A molecular weight of 76000 was estimated by gel filtration. The activity was heat sensitive but was not affected by hydrolytic enzymes except for pronase. The protein character of substance D91 was confirmed by gel electrophoresis and subsequent staining with Coomassie blue. The action exerted on sensitive bacteria was bacteriostatic rather than bactericidal. Biosynthesis of DNA, RNA, protein and polysaccharides were inhibited simultaneously in both Escherichia coli and Staphylococcus aureus. Active transport of glutamic acid was stopped in both S. cohnii and E. coli, whereas glucose uptake was inhibited in E. coli only. The substance induced a slow efflux of 86Rb+ from proloaded cells of S. cohnii and E. coli. The antagonistic activity of S. aureus D91 was eliminated by ethidium bromide at a rate of 47.6% suggesting that plasmids may be involved in its production.