While the importance of environmental analyses based on isotope discrimination has been growing, uncertainties remain about underlying phenomena. Published results on the temperature dependence of carbon isotope fractionation during methane oxidation in various media show different trends. A decrease in fractionation is generally expected with temperature, but some data for methane oxidation in aqueous media show an inverse relationship. This apparent contradiction was probed by representing the first methane oxidation step as three elementary processes: the adsorption of methane on the bacterial cell wall, the desorption of methane from the wall, and the conversion of methane into methanol mediated by methane monooxygenase (MMO) enzymes. Assuming that the proportion of vacant adsorption sites is stationary, a formula for the composite fractionation factor alpha was obtained. It was shown that alpha not only expresses the fractionation that may occur in each elementary process, but that it also depends on the ratio of the kinetic rates for conversion into methanol and desorption. This result and experimental data were used to estimate the activation energy for the desorption of methane from methanotroph cell wall in aqueous medium ( approximately 200 kJ/mol). Simple Rosso models of bacterial maximal-specific growth rate were then used to demonstrate that alpha and the isotope fractionation from the MMO-mediated conversion into methanol alone could vary in opposite ways as temperature changes, but that care must be exercised when using fitted relationships across wide temperature ranges.