Methane (CH4) is an important greenhouse gas that is produced in different subsurface environments. Its main biological sink, microbial CH4 oxidation, can be quantified in situ in the vadose zone using gas push-pull tests (GPPTs). This field method is based on the comparison of breakthrough curves of the reactant CH4 and a nonreactive tracer. Under diffusion-dominated transport conditions, previously employed noble gases are unsuitable as tracers to calculate rate constants for CH4 oxidation due to differing diffusion coefficients. Here, we show that by performing two consecutive GPPTs and coinjecting acetylene (C2H2) as an inhibitor of CH4 oxidation in the second test, the reactant CH4 can be used as a substitute tracer. Applying this procedure, apparent first-order rate constants for CH4 oxidation ranging from 0.38 to 0.82 h−1 were obtained in the vadose zone of three hummocks in an alpine peat bog near Lucerne, Switzerland. Corresponding estimates of in situ rates ranged from 4 to 299 ng CH4 g dry weight−1 h−1. In all but one GPPT, strong stable carbon isotope fractionation due to diffusion masked isotope fractionation due to microbial oxidation. Therefore, stable carbon isotope fractionation is suitable only to a limited extent as an indicator of microbial CH4 oxidation during a GPPT with diffusion-dominated gas transport. In contrast, the presented procedure for the quantification of microbial CH4 oxidation using GPPTs can be applied without restrictions even in systems with high porosity. Furthermore, the presented method may be useful for quantifying other processes for which suitable inhibitors but no suitable tracers are available.