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Dopamine beta-monooxygenase requires copper ions for catalytic activity. The stoichiometry of copper activation has been a matter of discussion, but most of the recent literature agrees on a model with two copper ions per active site. We have now reinvestigated this problem with kinetic experiments at high and low protein levels. The apoenzyme (metal free) is rapidly activated by adding copper. Incremental addition of copper to high levels (up to 10 microM subunits) of enzyme raised the catalytic activity until the stoichiometric relationship between copper and enzyme subunits was 1:1. No increase in activity was observed upon addition of copper in excess of this up to levels of 3 Cu/subunit. Experiments at low protein levels (0.12 microM subunits) revealed that copper activation is described by a hyperbolic, Michaelis-Menten-type curve. This is to be expected for the 1 Cu/subunit model, whereas the 2/1 model predicts sigmoid curves. With an incremental addition of apoenzyme (high level) to a fixed level of copper, a sharp break was again observed at 1 Cu/subunit, and excess apoenzyme showed no evidence of the inhibition predicted by the 2 Cu/subunit model. Steady-state kinetics experiments with variation of the concentrations of copper and the three substrates supported an equilibrium-ordered mechanism, whereby a single activating copper ion is trapped in the active site by the substrates. Treatment of enzyme containing more than 1 Cu/subunit [both the Cu(I) and Cu(II) states were examined] with a chelating column resulted in loss of all copper in excess of 1 Cu/subunit. Reactivation of apoenzyme by vanadyl ions was studied, both as dopamine beta-monooxygenase-catalyzed electron transfer and hydroxylation. The maximal velocity with vanadium was 70% of that with copper, and the activation curve was clearly hyperbolic, again supporting the requirement of only one metal ion per active site. In conclusion, our results support the view that the copper ions bound to dopamine beta-monooxygenase in excess of 1 Cu/subunit are not required for catalysis.