Cerulenin markedly inhibited the growth of Acholeplasma laidlawii. A. axanthum and A. granularum were less susceptible, whereas the sterol-requiring Mycoplasma species examined showed very little susceptibility. The inhibition was not reversed by the addition of long-chain fatty acids to the medium. At a concentration of 20 μg/ml, cerulenin inhibited the incorporation of [ 14 C]acetate into A. laidlawii membrane lipids, but it had no effect on either protein or nucleic acid biosynthesis. Cerulenin inhibited both the de novo synthesis of long-chain fatty acids and the elongation of medium-chain fatty acids. As a result, carotenoid biosynthesis was stimulated, and increased amounts of oleic and elaidic acids were incorporated into membrane polar lipids. Our studies support the concept that cerulenin can serve as a useful tool for obtaining better control of fatty acid composition of A. laidlawii membranes.