Tyrosine hydroxylase converts tyrosine to dihydroxyphenylalanine utilizing a tetrahydropterin cofactor and molecular oxygen. Previous deuterium isotope effect studies, have raised the possibility that the activation of oxygen might be the rate-limiting step for this reaction. To test the validity of this proposal, we have measured the O-18 kinetic isotope effects for the tyrosine hydroxylase reaction as a function of amino acid substrate, tetrahydropterin derivative, and pH. The measured O-18 isotope effects are nearly constant in every condition tested with an average value of 1.0175 +/- 0.0019. These results are consistent with a change in the bond order to oxygen in the rate determining step. Furthermore, the isotope effects measured with the coupled substrate 4-methoxyphenylalanine and the completely uncoupled substrate 4-aminophenylalanine are identical, implying the same rate determining step independent of whether oxygen activation is coupled to substrate hydroxylation. The results of these studies provide strong support for a rate limiting reductive activation of molecular oxygen, most likely via a one-electron transfer from the tetrahydropterin to form superoxide anion as the first reactive intermediate.