The kinetic properties of fatty acid synthetase, extracted from the mycelia of Neurospora crassa, were studied to determine the role of the multi-enzyme complex in the regulation of long chain saturated fatty acid synthesis. Acetyl-CoA, a substrate, affects the fatty acid synthetase with increasing concentration in a normal Michaelis-Menton kinetic mode. Malony-Coa, another substrate, activates the synthetase in a homotropic manner up to 35 microM with a Hill coefficient of 2.8. Above that concentration a sudden reverse trend in its effect takes over, then a gradual decrease in enzyme activity is observed with increasing substrate concentration. Citrate activates the synthetase to a maximum at 1 mM of citrate then reverses its effect and levels off at 2 mM. At the higher citrate concentrations the multi-enzyme complex has an activity twice that in the absence of citrate. Palmityl-CoA in low concentrations inhibits the synthetase. The inhibition is suppressed by the addition of bovine serum albumin. Increasing the concentration of the albumin enhances the suppression but does not eliminate the inhibition. These findings indicate that fatty acid synthetase could indeed have a key regulatory role.