Highly purified human-liver fatty acid synthetase complex was used to study the effect of several potential modifiers. Adenosine 3′,5′-phosphate did not alter the activity of either purified synthetase or of multienzyme present in 700 × g supernates. Its dibutyryl derivative was also ineffective when incubated with liver slices. Fructose 1,6-diphosphate, fructose 6-phosphate, and glucose 6-phosphate stimulated significantly the activity of the purified enzyme. Fructose 1,6-diphosphate, which was most effective, decreased the Km of the synthetase for NADPH. Phosphoenolpyruvate, rac-glycero-3 -phosphate and potassium phosphate were ineffective. All long-chain fatty acyl-CoA thioesters tested were inhibitory, but this effect was not observed until the regions of their critical micellar concentrations were reached. Free myristate, palmitate, and stearate did not inhibit synthetase activity up to the highest concentration tested (1 mM). An enzyme preparation derived from livers of fasted rats inactivated purified rat-liver 4′-phospho[14C]pantetheine-fatty acid synthetase by releasing its prosthetic group. It also decreased the activity of the purified human-liver complex.