The hepatic acid-soluble thiol content of striped mullet ( Mugil cephalus) exposed to cadmium (10 mg/liter seawater), mercury (200 μg/liter), fluorene (100 μg/liter), dibenzofuran (750 μg/liter), or a 20% water-soluble fraction of a No. 2 fuel oil, and of winter flounder ( Pseudopleuronectes americanus) exposed to pentachlorophenol (200 μg/liter) for up to 3 weeks was determined. Exposure to these chemicals caused an elevation of hepatic acid-soluble thiol content over control values. Similarly, the acid-soluble thiol content was 1.75 times control values 24 hr after injection with acetaminophen (40 g/100 g body wt, ip). In contrast, hepatic acid-soluble thiol concentrations did not fluctuate after feeding or during short-term starvation, or after acute or chronic physical trauma. Thus, the increase in hepatic acid-soluble thiols observed in mullet appears to be a specific response to chemical insult. The effect of chemical exposure on specific thiols can vary with the compound under investigation. Glutathione accounted for most of the acid-soluble thiol increase observed in mullet exposed to oil, whereas other acid-soluble thiols were also elevated after exposure to cadmium. Injection of [ 14C]glycine into cadmium- and oil-treated mullet showed that hepatic uptake of this amino acid substrate and its incorporation into hepatic glutathione increased after chemical exposure. The proportion of the total [ 14C]glycine in the liver incorporated into glutathione was unchanged after oil exposure, but was significantly increased in cadmium-exposed fish. These results suggest that chemicals may elevate glutathione content by enhancing the hepatic uptake of amino acid substrates and also the activity of biosynthetic enzymes.