Summary Aureofungin proved highly toxic to Saccharomyces cerevisiae and completely prevented the growth at a concentration of 1 µg/ml. The antibiotic (75 ng/ml) was most inhibitory to yeast growth at 15 to 20 °C than at 25 or 30-35 °C and in growth medium of pH 7 than of pH 3-5 or 8. Its antifungal action was reversed by addition of cholestrol to the medium while sulph-hydryl compounds (cysteine, reduced glutathione) and bivalent metal ions (Mg, Zn, Fe) had no effect. Aureofungin did not inhibit the incorporation of 14 C-leucine into protein, 14 C-uracil into RNA, and synthesis of DNA. However, it induced O 2 uptake and caused enormous leakage of 32 P from within the cells with corresponding high cellular mortality within a few hours of incubation. These effects were largely prevented when cholestrol was administered along with aureofungin into the reaction mixtures. It appears that antifungal action of aureofungin involves primarily a damage to cell membrane.