The effects of tetradecylthioacetic acid (TTA), insulin and dexamethasone on palmitoyl-CoA synthetase activity and its mRNA both in 7800 C1 hepatoma cells and cultured rat hepatocytes were studied. (1) When the hepatoma cells were cultivated in the presence of fatty acids or alkyl thioacetic acids (3-thia fatty acids) palmitoyl-CoA synthetase activity was increased several fold. The stronger effect was obtained with TTA, which also increased long-chain acyl-CoA synthetase mRNA significantly. TTA has no inducing effect on butyryl-CoA synthetase and little effect on octanoyl-CoA synthetase in the same cells. Dexamethasone also had inducing effect on palmitoyl-CoA synthetase in the hepatoma cells. Insulin counteracted the induction given by TTA. All of these regulation actions take place at the pretranslational level. (2) In isolated hepatocytes the activity of palmitoyl-CoA synthetase was much higher than in hepatoma cells, but it was lost rapidly in culture. The loss of the enzyme activity was slowed down in the presence of TTA and insulin, either alone or combined. Dexamethasone combined with TTA reversed the loss of enzyme activity, while dexamethasone alone even increased the loss. Analysis of palmitoyl-CoA synthetase mRNA shows that TTA prevents the loss of the enzyme activity by inducing mRNA of the enzyme, dexamethasone enhances the effect of TTA, while insulin stabilizes the enzyme activity in the cultured cells without increasing the mRNA level.