Cerulenin, an inhibitor of fatty acid synthesis, specifically inhibits the biosynthesis of leucomycin, a 16-membered macrolide antibiotic, in both growing cells and resting cells of Streptomyces kitasatoensis. In growing cells, the production of leucomycin was inhibited as long as cerulenin remained in the culture. In resting cells, 50 percent inhibition was achieved with a cerulenin concentration of 1.5 microgram/ml. Cells in which leucomycin synthesis was inhibited for 9 h remained capable of leucomycin synthesis upon removal of the inhibitor. Cerulenin specifically inhibits the incorporation of [14C]acetate into leucomycin but does not affect total protein or RNA synthesis. The uptake of [14C]acetate was not inhibited under conditions which completely inhibited the incorporation of acetate into leucomycin. Since cerulenin is known to block the condensation of malonyl-CoA subunits in the formation of fatty acids, it can be concluded that the aglycone of leucomycin is synthesized via the polyketide pathway by condensation steps similar to those involved in fatty acid biosynthesis.