Nickel, a potentially toxic metal, is present in all soils with an average concentration of 20 to 30 mg/kg, sometimes exceeding 10,000 mg/kg (e.g., ultramafic soils). The ecotoxicological risk of Ni in soils to organisms is controlled by its availability. It is therefore essential to identify an efficient and reliable method for the evaluation of this risk. This paper presents a complete study of the effect of Ni origin, localization, and soil properties on its availability as assessed with the isotopic exchange kinetics (IEK) method and compares plant response to isotopically exchangeable properties of Ni in soils. We performed IEK on 100 soil samples representing a worldwide range of Ni fate, and concentrations showed that pH was the main influencing parameter and that labile Ni (i.e., isotopically exchangeable Ni, Et) could be reasonably well assessed by a single diethylene triamine pentaacetic acid extraction. The identification of the soil mineral phases that bear Ni (bearing phases) in 16 Ni-rich samples selected among the 100 soils showed a strong effect of the mineralogy of the bearing phases on Ni availability (IEK). Plants with different Ni accumulation strategies all took up Ni from the same labile pool of Ni in four contrasting soils, and the amount taken up by hyperaccumulator plants could be anticipated with the IEK parameters, thus confirming the usefulness of isotopic dilution methods for risk assessment.