Sporulation of Saccharomyces cerevisiae G2-2 was inhibited by the antibiotic cerulenin which is known to be a specific inhibitor of fatty acid and sterol synthesis. This inhibition was reversed by various fatty acids, especially by oleic acid (C(18:1)) and pentadecanoic acid (C(15:0)). Ergosterol showed only slight reversibility of this inhibition. When cerulenin was added to the sporulation medium later than 12 h after the start of incubation, the marked inhibition disappeared. When the fatty acid fraction extracted from the sporulated yeasts was added to the cells pretreated with cerulenin for more than 6 h, sporulation became evident 6 h after the fatty acid fraction addition. Therefore, sufficient synthesis of fatty acids required for sporulation was assumed to occur during the first 6 h in phase I of yeast sporulation.