The pharmacokinetics of the spin-trap alpha-phenyl-N-tert-butylnitrone (PBN) was investigated in male Sprague-Dawley rats. Plasma concentrations after i.v. administration (10 mg/kg) declined monoexponentially with a terminal half-life of 2.01 +/- 0.35 h and total plasma clearance (CL(p)) and volume of distribution at steady state (Vd(ss)) averaged 12.37 +/- 3.82 ml/min/kg and 1.74 +/- 0.5 l/kg, respectively. The observed CL(p) was in close agreement with the blood clearance (CL(b)) value (11.5 ml/min/kg) predicted from in vitro liver microsomal incubations suggesting that PBN CL(p) in rats is predominantly due to hepatic metabolism. Peak plasma concentration (C(max)) following p.o. (20 mg/kg) and s.c. (30 mg/kg) PBN administration was 7.35 +/- 1.92 and 3.56 +/- 0.66 microg/ml, whereas the area under the concentration-time curve from 0 to infinity was 23.89 +/- 5.84 and 15.96 +/- 3.10 microg-h/ml, respectively. The mean oral bioavailability of PBN was 85.63 +/- 20.93%. Biotransformation studies indicated the P450 2C11-catalyzed hydroxylation of PBN to M1. Potential sites of hydroxylation included the benzylic carbon resulting in phenyl-N-tert-butylhydroxamic acid or the phenyl ring that would afford alpha-hydroxyphenyl-N-tert-butylnitrone (HOPBN). The structure of M1 was established as alpha-4-Hydroxyphenyl-N-tert-butylnitrone (4-HOPBN) on the basis of: 1) obvious LC R(t) differences between M1 and the authentic hydroxamate standard, 2) P450 catalyzed hydroxylation of [(2)H]PBN that contained a deuterium instead of a hydrogen atom on its benzylic position and which afforded [(2)H]M1, and 3) comparison of the liquid chromatography-tandem mass spectrometry properties with a synthetic 4-HOPBN standard. We speculate that 4-HOPBN is an "active" PBN metabolite that provides an additive effect to the pharmacological action of PBN in vivo.