The kinetics of the reaction of acetyl coenzyme A (AcCoA) with fatty acid synthase has been studied with a modified quenched-flow technique in 0.1 M potassium phosphate (pH 7.0), 0.5 mM ethylenediaminetetraacetic acid, and 10% glycerol (w/v) at 23 degrees C. The kinetics of the deacetylation of the isolated acetylated enzyme by CoA also was studied. An overall mechanism consistent with the data is (formula; see text) where E represents the enzyme. The equilibrium dissociation constants, K1 and K3, were estimated to be 85 and 70 microM, respectively, and the rate constants k2 and k-2 are 43 and 103 s-1, respectively. The maximum number of acetyl groups bound to the enzyme in terms of this mechanism is 3.8 (mol/mol). This mechanism also is consistent with the amount of acetylated enzyme formed during titrations of the enzyme and radioactive AcCoA with CoA. The spontaneous hydrolysis of the enzyme at 23 degrees C has a rate constant of 4.7 X 10(-4) s-1. The acetyl groups on the native enzyme are rapidly removed by hydroxylamine. However, 0.39 of the acetyl groups remains after treatment with hydroxylamine if the enzyme is first denatured in 4 M urea. This suggests that the acyl binding sites on the native enzyme are an unstable acetyl oxygen ester and an acetyl thio ester. Destruction of the thioesterase activity of the enzyme through chemical modification of the enzyme does not alter the rate of spontaneous hydrolysis of the acetyl-enzyme nor its reactivity toward hydroxylamine.