Mechanistic studies on the aliphatic ligand hydroxylation in a copper complex of tridentate ligand 1a {N,N-bis[2-(2-pyridyl)ethyl]-2-phenylethylamine} by O-2 have been performed in order to shed light on the structure and reactivity of the active oxygen species of our functional model for copper monooxygenases (Itoh, S.; et al. J. Am. Chem. Soc. 1995, 117, 4714). When the copper complex [Cu-II(1a)(ClO4)(2)] was treated with an equimolar amount of benzoin and triethylamine in CH2Cl2 under O-2 atmosphere, efficient hydroxylation occurred selectively at the benzylic position of the ligand to provide oxygenated product 2a {N,N-bis[2(2-pyridyl)ethyl]-2-phenyl-2-hydroxyethylamine} quantitatively. An isotope labeling experiment using O-18(2) confirms that the oxygen atom of the OH group in 2a originates from molecular oxygen. Spectroscopic analyses using UV-vis, resonance Raman, and ESR on the reaction of [Cu-I(1a)](+) and O-2 at low temperature show that a mu-eta(2):eta(2)-peroxodicopper(II) complex is an initially formed intermediate. Kinetic analysis on the peroxo complex formation indicates that the reaction of the Cu(I) complex and the monomeric superoxocopper(II) species is rate-determining for the formation of the mu-eta(2):eta(2)-peroxodicopper(II) intermediate. When ligand 1a is replaced by 1,1,2,2-tetradeuterated phenethylamine derivative 1a-d(4), a relatively small kinetic deuterium isotope effect (k(H)/k(D) = 1.8 at -40 degrees C) is observed for the ligand hydroxylation step. The rate of the hydroxylation step is rather insensitive to the p-substituent of the ligand [(PyCH2CH2)(2)NCH2CH2Ar, 1a Ar = C6H5; 1b Ar = p-CH3C6H4, 1c Ar = p-ClC6H4, and 1d Ar = p-NO2C6H4)], but it varies depending on the solvent (THF > acetone > CH3OH > CH2Cl2). The p-substituent, the solvent, and the kinetic deuterium isotope effects suggest that O-O bond homolysis of the mu-eta(2):eta(2)-peroxodicopper(II) intermediate is involved as a rate-determining step in the aliphatic ligand hydroxylation process. Based on the results of the kinetics and the crossover experiments, we propose a mechanism involving intramolecular C-H bond activation in a bis-mu-oxodicopper(III)) type intermediate for the ligand hydroxylation reaction.