Availability is a key property for the assessment of soil-to-plant transfer of heavy metals. This work was conducted to determine whether the available pool of Ni differs according to the ability of plants to take up and accumulate the metal. An excluder plant species (Triticum aestivum L.), an indicator (Trifolium pratense L.), and 3 populations of the Ni-hyperaccumulator Alyssum murale (Waldst. & Kit.) were grown for 90 days on 4 soils with a gradient of concentrations of total and available Ni. Isotopic exchange methods with ⁶³Ni ions were used to measure the exchangeable soil Ni (E-value, intensity, and capacity factors), to monitor its uptake by plants and to determine the size of the available pool (L-value). Results showed that, for a given soil, the L-values were similar for all plant species, showing that they all access the same Ni exchangeable pool regardless of their Ni uptake capacity. Also, L-values for a given soil were equal to the E-value calculated for a 90-day period, demonstrating that plant Ni originated from the isotopically exchangeable soil Ni. This pool can be accurately and simply determined with the isotopic exchange kinetic methods run on soil–solution batch systems without plants. Moreover, the results indicate that the plant species take up Ni as a response to ‘intensity’, ‘capacity’, and ‘quantity’ soil factors and that E-value alone is not enough to predict plant uptake. This work suggests a uniform behaviour of the plants tested towards soil Ni and may have practical applications in phytoextraction and phytomining, as the plants removed Ni exclusively from the exchangeable pool.