The effect of different fatty acids on cytochrome P4504A1 mRNA levels was studied in primary cultures of rat hepatocytes, using a solution hybridization assay. All fatty acids tested induced P4504A1 mRNA levels in a dose- and time-dependent manner. Most potent were docosahexaenoic acid (22:6) and arachidonic acid (20:4), both of which gave a 6-fold increase in mRNA levels at 300 microM, followed by linolenic acid (18:3) and lauric acid (12:0). The effect of three different sulfur-substituted fatty acids was investigated. Tetradecylthioacetic acid, which is blocked for beta-oxidation by sulfur substitution of the beta-carbon, induced P4504A1 mRNA levels 8-fold at 300 microM concentration, whereas tetradecylthiopropionic acid, which can undergo one round of beta-oxidation, only gave a 2-fold increase at the same concentration. The most pronounced effect was seen with 3,14-dithiahexadecanedioic acid, a dicarboxylic acid with both beta-carbons blocked for beta-oxidation, which gave a 31-fold induction of mRNA levels at 300 microM. In time-course studies the effect of docosahexaenoic acid, 3,14-dithiahexadecanedioic acid and the potent peroxisomal proliferator Wy 14,643 on P4504A1 mRNA levels was already detectable after 2 h and maximal after 48 h of treatment, which was reflected in increased levels of P4504A1 protein. Taken together, these results show that endogenous fatty acids, such as docosahexaenoic acid and arachidonic acid, act as pretranslational regulators of P4504A1 when added to primary cultures of rat hepatocytes. Blocking their metabolism (beta-oxidation) leads to significant enhancement of their activity.