The 18O content of atmospheric O2 is an important tracer for past changes in the biosphere. Its quantitative use depends on knowledge of the discrimination against 18O associated with the various O2 consumption processes. Here we evaluated, for the first time, the in situ 18O discrimination associated with soil respiration in natural ecosystems. The discrimination was estimated from the measured [O2] and δ18O of O2 in the soil-air. The discriminations that were found are 10.1 ± 1.5‰, 17.8 ± 1.0‰, and 22.5 ± 3.6‰, for tropical, temperate, and boreal forests, respectively, 17.9 ± 2.5‰ for Mediterranean woodland, and 15.4 ± 1.6‰ for tropical shrub land. Current understanding of the isotopic composition of atmospheric O2 is based on the assumption that the magnitude of the fractionation in soil respiration is identical to that of dark respiration through the cytochrome pathway alone (∼18‰). The discrimination we found in the tropical sites is significantly lower, and is explained by slow diffusion in soil aggregates and root tissues that limits the O2 concentration in the consumption sites. The high discrimination in the boreal sites may be the result of high engagement of the alternative oxidase pathway (AOX), which has high discrimination associated with it (∼27‰). The intermediate discrimination (∼18‰) in the temperate and Mediterranean sites can be explained by the opposing effects of AOX and diffusion limitation that cancel out. Since soil respiration is a major component of the global oxygen uptake, the contribution of large variations in the discrimination, observed here, to the global Dole Effect should be considered in global scale studies.