Biotransformation of phenacetin via O-deethylation to acetaminophen, an index reaction reflecting activity of Cytochrome P450-1A2, was studied in microsomal preparations from a series of human livers. Acetaminophen formation was consistent with a double Michaelis-Menten system, with low-Km (mean Km1 = 68 microM) and high-Km (mean Km2 = 7691 microM) components. The low-K(m) enzyme accounted for an average of 96% of estimated intrinsic clearance, and was predicted to contribute more than 50% of net reaction velocity at phenacetin concentrations less than 2000 microM. Among index inhibitor probes, alpha-naphthoflavone was a highly potent inhibitor of the low-Km enzyme (Ki1 = 0.013 microM); furafylline also was a moderately active inhibitor (Ki1 = 4.4 microM), but its inhibiting potency was increased by preincubation with microsomes. Ketoconazole was a relatively weak inhibitor (Ki1 = 32 microM); quinidine and cimetidine showed minimal inhibiting activity. Among six selective serotonin reuptake inhibitor (SSRI) antidepressants, fluvoxamine was a potent inhibitor of 1A2 (mean Ki1 = 0.24 microM). The other SSRIs were more than tenfold less potent. Mean Ki1 values were: fluoxetine, 4.4 microM; norfluoxetine, 15.9 microM; sertraline, 8.8 microM; desmethylsertraline, 9.5 microM; paroxetine, 5.5 microM. The antidepressant nefazodone and four of its metabolites (meta-chloro-phenylpiperazine, two hydroxylated derivatives, and a triazoledione) were very weak inhibitors of P450-1A2. Venlafaxine and its O- and N-desmethyl metabolites showed minimal inhibitory activity.