Phenacetin is metabolized primarily by O-deethylation to paracetamol (POD activity), a reaction catalysed by cytochrome P450. The high affinity component of POD activity is inducible in rat liver by treatment of the animals with polycyclic aromatic hydrocarbons. Following treatment with hydrocarbons such as 3-methylcholanthrene (MC) and isosafrole (ISF) both cytochromes P450c (P450IA1) and P450d (P450IA2) are also induced in rat liver. Studies with the reconstituted enzymes have shown that both forms of P450 catalyse phenacetin O-deethylation at rates that exceeded that of the high affinity component of activity of hepatic microsomal preparations from 3-methylcholanthrene-treated rats (at 4 microM phenacetin: P450c, 440 +/- 40 pmol/nmol/min; P450d, 1030 +/- 10 pmol/nmol/min; microsomal fraction, 163 pmol/mg/min). Specific inhibitory antibodies (both monoclonal and monospecific polyclonal) were used to define the specificity of microsomal POD activity. These studies have shown that hepatic high affinity POD activity is exclusively catalysed by cytochrome P450d in both untreated rats and in rats pretreated with MC.