Dopamine beta-hydroxylase (EC 1.14.17.1) is inactivated by p-hydroxybenzylcyanide (PHBC) in a manner characteristic of a suicide substrate. The inactivation 1) is first order in inhibitor (Kd = 1.9 mM, k2 = 0.05 min-1, pH 5.0), 2) exhibits saturation kinetics, and 3) is dependent on O2 and ascorbate. Restoration of activity could not be achieved by dialysis. The substrate, p-tyramine, protects the enzyme from inactivation, while in initial velocity kinetic experiments, PHBC is a linear competitive inhibitor (Kis = 2.6 mM) versus p-tyramine. The Kis value for PHBC is in good agreement with the Kd value obtained from analysis of the inactivation reaction. PHBC (in the presence of O2 and ascorbate) is also a substrate for the enzyme, being converted to p-hydroxymandelonitrile (PHMN) at a relative Vmax 13% that of p-tyramine. Under these conditions, the ratio of product formation to inactivation is 8000:1, suggesting that PHMN (or a tautomer of PHMN) is the species responsible for inactivation. Indeed, incubation of the enzyme with PHMN in the absence of ascorbate leads to irreversible inactivation. Since PHMN breaks down to p-hydroxybenzaldehyde and cyanide, experiments were conducted to determine whether these compounds may have been responsible for the inactivation. Incubation of the enzyme with p-hydroxybenzaldehyde did not lead to inactivation, whereas the inactivation produced with cyanide could be reversed by dialysis. Thus, the data point to PHMN as the molecule responsible for time-dependent loss of activity of the enzyme. Together, these experiments demonstrate that the newly discovered inhibitor, PHBC, meets the critieria for a suicide substrate for dopamine beta-hydroxylase.