Abstract: Hydroxylation and N-methylation of three potent α2-adrenoceptor agonists detomidine, medetomidine and dexmedetomidine by rat brain, kidney, liver and lung were studied in vitro. NADPH-dependent hydroxylation of the 3′-methyl group was catalyzed mainly by the microsomal fraction of liver. Monooxygenation by the other tissues was negligible. The hepatic monooxygenase reaction was characterized by high affinity (Km 5.5-9.8 μM) and low capacity (Vmax 29-66 pmol/min. per mg protein). Statistically significant (P < 0.05) differences between the Km and Vmax values of medetomidine and dexmedetomidine were observed suggesting stereoselective oxidation of the drug molecule. N-Methylation of the arylalkylimidazoles occurred mainly in the cytosolic fraction of the kidney. With S-adenosylmethionine as the methyl donor, the transferase activities in other tissues were less than 10% of the specific activity of the renal enzyme (about 7 pmol/min. per mg protein for medetomidine). Even the renal enzyme showed a relatively low capacity (Km 250-300 μM and Vmax 15-33 pmol/min. per mg protein). Different tissue distributions of the arylalkylimidazole N-methyltransferase and histamine N-methyltransferase suggest that they are two distinct enzyme species. Based on these results, aliphatic hydroxylation is the major (metabolic) elimination mechanism of the arylalkylimidazole drugs, and liver is the principal eliminating organ. The importance of N-methylation is diminished by its low capacity and probable regeneration of the parent drug by demethylation.