The stoichiometry of the copper requirement for the dopamine beta-monooxygenase-catalyzed conversion of dopamine to norepinephrine has been investigated by rapid chemical-quench techniques. This approach, which employs concentrated samples of enzyme, overcomes ambiguities of interpretation arising from levels of trace copper in excess of enzyme concentrations normally added to steady state kinetic assays. Low turnover numbers are observed when rapid quench kinetic studies are performed under conditions in which enzyme concentrations (2.5-7.1 microM) are in excess over trace copper levels (about 0.7 microM). The addition of exogenous Cu(II) results in full restoration of activity, which is maximal at a stoichiometry of 2 mol of copper/mol of enzyme subunit. From the dependence of catalytic activity on copper levels we conclude that both coppers are required for catalysis. No stimulation of activity was observed upon addition of the following metal ions: Ni(II), Co(II), Mn(II), Fe(III), and Zn(II). In addition, the magnitude of the tritium isotope effect for [2-3H]dopamine hydroxylation is invariant over a large range of enzyme activities accompanying changes in the ratio of copper to enzyme concentration. These results appear to rule out an effector role for the second mole of copper/subunit, implicating both copper atoms in active site redox chemistry.