1,8-Cineole, the monoterpene cyclic ether known as eucalyptol, is one of the components in essential oils from Eucalyptus polybractea. We investigated the metabolism of 1,8-cineole by liver microsomes of rats and humans and by recombinant cytochrome P450 (P450 or CYP) enzymes in insect cells in which human P450 and NADPH-P450 reductase cDNAs had been introduced. 1,8-Cineole was found to be oxidized at high rates to 2-exo-hydroxy-1,8-cineole by rat and human liver microsomal P450 enzymes. In rats, pregenolone-16alpha-carbonitrile (PCN) and phenobarbital induced the 1,8-cineole 2-hydroxylation activities by liver microsomes. Several lines of evidence suggested that CYP3A4 is a major enzyme involved in the oxidation of 1,8-cineole by human liver microsomes: (1), 1,8-cineole 2-hydroxylation activities by liver microsomes were inhibited very significantly by ketoconazole, a CYP3A inhibitor, and anti-CYP3A4 immunoglobulin G; (2), there was a good correlation between CYP3A4 contents and 1,8-cineole 2-hydroxylation activities in liver microsomes of eighteen human samples; and (3), of various recombinant human P450 enzymes examined, CYP3A4 had the highest activities for 1,8-cineole 2-hydroxylation; the rate catalyzed by CYP3A5 was about one-fourth of that catalyzed by CYP3A4. Kinetic analysis showed that K(m) and V(max) values for the oxidation of 1,8-cineole by liver microsomes of human sample HL-104 and rats treated with PCN were 50 microM and 91 nmol/min/nmol P450 and 20 microM and 12 nmol/min/nmol P450, respectively. The rates observed using human liver microsomes and recombinant CYP3A4 were very high among other CYP3A4 substrates reported so far. These results suggest that 1,8-cineole, a monoterpenoid present in nature, is one of the effective substrates for CYP3A enzymes in rat and human liver microsomes.