Cerulenin, an antibiotic with the structure of (2R)(3S)-2,3-epoxy-4-oxo-7,10-dodecadienoylamide, irreversibly inactivates yeast fatty acid synthetase. Of all catalytic activities of the synthetase, only the condensation reaction is inhibited by cerulenin. At 0 degrees C and pH 6.5, the second-order rate constant of k = 88 M-1 . S-1 was obtained for the inactivation by cerulenin. This value was about 90-times greater than the rate constant for the inactivation of the enzyme by iodoacetamide. The enzyme was protected against the action of cerulenin by prior treatment with acetyl-CoA but not malonyl-CoA. Treatment of the enzyme with iodoacetamide, while impairing the synthetase activity, induced malonyl-CoA decarboxylase activity [Kresze, G.-B., Steber, L., Oesterhelt, D., and Lynen, F. (1977) Eur. J. Biochem. 79, 191-199]. Cerulenin had no effect on the malonyl-CoA decarboxylase activity of the iodoacetamide-treated enzyme. N-Ethylmaleimide, in contrast, inhibited the iodoacetamide-induced malonyl-CoA decarboxylase activity. When the enzyme was preincubated with cerulenin, malonyl-CoA decarboxylase activity could not be detected even after treatment of the enzyme with iodoacetamide. These results indicated that the reaction of cerulenin with the peripheral SH-groups of the synthetase is responsible for the inactivation.