The electronic and steric constraints of the dopamine beta-monooxygenase (DbetaM; E.C. 1.14.17.1) active site were studied using a series of chiral bisubstrate inhibitors. The (R) and (S) enantiomers of 5-phenyl-2-thiooxazolidone were apparent bisubstrate inhibitors for DbetaM with respect to tyramine and dioxygen, but with small enantiomeric selectivity. In contrast to the substrate specificity of the enzyme, N-methylation of both inhibitors increased the potency without altering the enantiomeric selectivity. The (S) C-4-methyl substitution was more detrimental toward the inhibition potency compared to (R) C-4-methyl substitution for both the (R) and (S) series, which was also opposite of the substrate specificity of the enzyme. The high inhibition potency and apparent bisubstrate behavior of 3-phenyl-1,5-bisthioglutarimide (XVI), a probe designed to mimic two distinct binding modes for the (R) and (S) inhibitors, suggested that they may interact with the enzyme by two different modes involving both coppers in the active site. Direct support for the interaction of the thione group(s) of XVI with the reduced DbetaM copper(s) is provided by the UV-vis spectroscopic studies. The complete disappearance of the characteristic UV absorption of XVI at 336 nm in the presence of stoichiometric amounts of reduced DbetaM demonstrate that it could be an active site titrant for reduced DbetaM. The ability of the enzyme to interact with these inhibitors by more than one mode suggests that the DbetaM active site possesses high steric and electronic tolerance.