(1) The activities of peroxisomal beta-oxidation and palmitoyl-CoA hydrolase in Morris hepatoma 7800 C1 cells were studied. The cells were grown until they reached steady state (constant DNA content per dish) and then were cultured in the presence of fatty acids or alkylthioacetic acids, i.e., S-substituted fatty acid analogues. (2) The fatty acid analogues increased the activity of the cyanide-insensitive palmitoyl-CoA oxidase several-fold. The effect was dose-dependent; 5 microM tetradecylthioacetic acid (TTA) was sufficient to give a significant induction. With 20 microM TTA, the increase in enzyme activity was discernable after 3 h and reached a maximum after 3 days. The inducing effect of the alkylthioacetic acids increased with the length of the hydrophobic alkyl end of the analogue. The inducing ability disappeared when the fatty acid analogue was omega-oxidized to the corresponding dicarboxylic acid. Oxidation of the sulfur atom resulted in inhibited cellular uptake and abolished enzyme induction. (3) At higher concentrations (0.5-1 mM), normal fatty acids also induced cyanide-insensitive palmitoyl-CoA oxidation. Myristic acid was the most potent inducer, whereas fatty acids with shorter as well as longer carbon chains were less efficient. The inducing effect increased with the number of double bounds in the fatty acid. (4) The normal fatty acids as well as the fatty acid analogues also induced palmitoyl-CoA hydrolase, but the relative changes were much less pronounced than with the palmitoyl-CoA oxidase.