The chemistry and energetics of the initial C-H bond cleavage step in dopamine beta-monooxygenase (D beta M, E.C. 1.14.17.1) catalysis has been explored using 1-(2-aminoethyl)-1,4-cyclohexadiene (CHDEA) as a probe. D beta M has been previously shown (Wimalasena, K.; May, S. W. J. Am. Chem. Soc. 1989, 111, 2729) to catalyze the aromatization of CHDEA to phenylethylamine (PEA). We now report that the side chain hydroxylated product, 2-amino-1-(1,4-cyclohexadiene)ethanol (CHDEA-OH), is also a direct product of the D beta M/CHDEA reaction. The PEA:CHDEA-OH product ratio is 2.7 at pH 5.2 and 37 degrees C for the D beta M/CHDEA reaction. The side chain deuterated analog, 1-(2-amino-1,1-dideuterioethyl)-1,4-cylohexadiene, undergoes aromatization almost exclusively. The ring deuterated analog, 1-(2-aminoethyl)-3,3,6,6-tetradeuterio-1,4-cyclohexadiene, favors side chain hydroxylation 2.6 times over aromatization. The hexadeuterio derivative, 1-(2-amino-1,1-dideuterioethyl)3,3,6,6-tetradeuterio-1,4-cyclohexadiene (CHDEA-d(6)), prefers aromatization 3.9 times. The apparent kinetic isotope effects of CHDEA-d(6) are small, 1.8 for k(cat) and 1.3 for k(cat)/K-m, suggesting that the initial C-H bond cleavage is only partly rate limiting in the CHDEA/D beta M reaction analogous to the normal D beta M hydroxylation reaction. The intrinsic isotope effects for the exocyclic and ring methylene hydrogens are estimated to be 9.9 and 7.0, respectively. The alteration of the product ratio due to deuterium substitution suggests that activation energies of the initial C-H bond cleavage steps for the two pathways are similar. In contrast to the high thermodynamic driving force for the ring methylene hydrogen abstraction, the similar activation energies must be a consequence of the proximity and/or the relative orientation of the corresponding hydrogens of the enzyme-bound CHDEA with respect to the activated copper oxygen species.