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Carbon isotope fractionation factors reported for aerobic bacterial oxidation of CH₄ [Formula: see text] range from 1.003 to 1.039. In a series of experiments designed to monitor changes in the carbon isotopic fractionation of CH₄ by Type I and Type II methanotrophic bacteria, we found that the magnitude of fractionation was largely due to the first oxidation step catalyzed by methane monooxygenase (MMO). The most important factor that modulates the [Formula: see text] is the fraction of the total CH₄ oxidized per unit time, which strongly correlates to the cell density of the growth cultures under constant flow conditions. At cell densities of less than 0.1g/L, fractionation factors greater than 1.03 were observed, whereas at cell densities greater than 0.5g/L the fractionation factors decreased to as low as 1.002. At low cell densities, low concentrations of MMO limit the amount of CH₄ oxidized, while at higher cell …