Variations in the Dole Effect, which have been used to infer past changes in biospheric productivity, are strongly affected by isotopic discrimination in soil respiration. Respiration through the alternative oxidase (AOX) pathway is associated with a higher discrimination than the one associated with the “normal” dark respiration pathway (the cytochrome pathway, COX). However, observations of O2 discrimination and AOX activity in undisturbed natural environments are scarce. In the current study we measured the O2 concentration and stable isotopes in the root zone of tundra, boreal forest and alpine forest soils. To estimate the discrimination from this data, we have performed O2diffusion experiments in gamma-sterilized soil columns, with varying soil clay content. The discrimination found in the diffusion experiments was independent of clay content, and the value found, 14 ± 2‰, is the same as the one for binary diffusion of O2 in N2, indicating no interaction between the O2 and clay particles. Based on the field and laboratory results, the respiratory discrimination in the soils studied is 15–31‰, with the higher values associated with colder soils. The high discrimination found for cold (<6°C) soils indicates that AOX is a major respiratory pathway in these soils. This relationship between soil temperature and discrimination can be used in future interpretations of Dole Effect variations.