The reduced hydroxylase component (MMOH) of soluble methane monooxygenase (MMO) from Methylosinus trichosporium OB3b reacts with O2 and CH4 to produce CH3OH and H2O in a single-turnover reaction. Transient kinetic analysis of this reaction has revealed at least five and probably six intermediates during the turnover [Lee, S.-K., Nesheim, J. C., and Lipscomb, J. D. (1993) J. Biol. Chem. 268, 21569-21577; Liu, Y., Nesheim, J. C., Lee, S.-K., and Lipscomb, J. D. (1995) J. Biol. Chem. 270, 24662-24665]. One intermediate, termed compound Q, reacts with CH4 to yield enzyme-bound product. It is shown here that the deuterium kinetic isotope effect (KIE) for the reaction of compound Q with CH4 is 50-100, which is one of the largest effects observed to date. The rate constants for the reactions of the deuterated homologs of methane decrease monotonically as the deuterium content increases, suggesting that a large primary isotope effect dominates. The KIEs determined by analyzing the products after a single turnover have the following values: 1:1 CH4:CD4 (19); CD3H (12); CD2H2 (9); and CH3D (4). The KIE values determined by directly observing the reactive intermediate and by monitoring product ratios are all large, consistent with complete C-H bond breaking in the oxygenation step of the reaction. However, the differences in the KIE values determined by these two methods suggest that the reaction is more complex than currently proposed. A modified mechanism introducing the possibility of hydrogen-atom reabstraction by an intermediate methyl radical is proposed.