Metabolism of acetaminophen (APAP) to its reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI) is mediated by cytochrome P450. A pharmacokinetic study was conducted to quantitate changes in the formation clearance (Cl(f)) of NAPQI to assess in vivo the activation and inhibition of NAPQI formation by methylxanthines. Cl(f) of NAPQI was unaltered by methylxanthine administration in saline-pretreated rats. In phenobarbital-induced rats receiving a nontoxic dose of APAP (100 mg/kg i.v.), a single dose of caffeine (100 mg/kg i.p.) co-administered with APAP increased the Cl(f) of NAPQI formation from 0.58 +/- 0.47 to 2.08 +/- 1.1 1 ml/min/kg (P = .01). Unlike caffeine, theophylline (93 mg/kg i.p.) had no effect on the Cl(f) of NAPQI in phenobarbital-induced rats. The increase in the Cl(f) of NAPQI immediately after a single dose of caffeine demonstrates that P450 activation by caffeine can occur in vivo, as we observed previously in microsomes. The same dose of APAP and methylxanthines also was administered to rats induced with methylcholanthrene. The co-administration of either a single dose of caffeine or theophylline diminished the Cl(f) of NAPQI by 86% (P = .01) and 52% (P = .03), respectively. These in vivo results agree with our previous studies of the effects of the methylxanthines on the formation of NAPQI in rat liver microsomes.