The steady-state kinetics of chicken liver fatty acid synthase has been studied over the pH range 5.9-8.6 in 0.1 M potassium phosphate/1 mM EDTA at 25.0 degrees C. The steady-state initial velocity, v, which was determined by measuring the rate of consumption of NADPH spectrophotometrically over a wide range of substrate concentrations, followed the rate law v = (formula; see text), in which Ac-CoA is acetyl-CoA, Mal-CoA is malonyl-CoA, the Kj are Michaelis constants, the Kj,i are inhibition constants, kcat is the turnover number, and [E0] is the total enzyme concentration. The product CoA is an inhibitor at high concentrations but activates the enzyme at low concentrations when the concentration of Ac-CoA is high. The rate law can be derived from a simple multistep mechanism; in terms of this mechanism, the Michaelis constants are lower bounds to the substrate dissociation constants, and the turnover number contains the first-order rate constants characterizing the reactions required to produce palmitic acid. Plots of kcat, kcat/KN, kcat/KA, and kcat/KM versus pH are bell shaped. Analysis of the results in terms of two ionizable groups indicates that in all cases an ionizable group with an apparent pKa of approximately equal to 6 is of importance. For kcat and kcat/KN, the apparent pKa of the second ionizable group is approximately equal to 7.8, whereas for kcat/KA and kcat/KM, it is approximately equal to 7.