We propose a highly sensitive method for the measurement of catechol-O-methyltransferase (COMT) activity with norepinephrine (NE), an endogenous native substrate. The product, normetanephrine, was determined by high-performance liquid chromatography (HPLC)-peroxyoxalate chemiluminescence reaction detection or, if required, less sensitive fluorescence detection. For the measurement of membrane-bound (MB)-COMT activity in the rat erythrocyte, the HPLC-peroxyoxalate chemiluminescence reaction detection was employed. Soluble (S)- and MB-COMT activities in the rat erythrocyte were 22.9 ± 2.5 and 4.62 ± 1.23 pmol min⁻¹ (mg protein)⁻¹, respectively (n = 5). The K_m values obtained for S- and MB-COMT were 366 ± 31 μmol l⁻¹ and 12.0 ± 1.1 μmol l⁻¹, respectively (n = 5), suggesting that the use of NE as a substrate would give more precise information on the role of both isoenzymes. However, with dihydroxybenzoic acid as an artificial substrate, the K_m values for S- and MB-COMT were similar, with values of 69.2 ± 11.4 μmol l⁻¹ and 72.2 ± 9.2 μmol l⁻¹, respectively. The proposed method is thought to be useful for the measurement of both S-COMT and MB-COMT activities, and would give us critical information on the role of metabolism of catecholamines in rat tissues.