We investigated the speciation of Ni in organic and mineral smelter contaminated soils that had been previously treated with lime and evaluated the effect soil type and treatment on Ni speciation and plant availability. Using a combination of macroscopic and microscopic techniques including SEM, and micro-x-ray absorption fine structure (XAFS) and x-ray fluorescence (XRF) spectroscopies, we found that NiO dominated the Ni speciation in both soils; with Ni layered double hydroxide (LDH) precipitates, and organically complexed Ni making up the latter fractions in the loam and muck soils respectively. Metal hyperaccumulating plants have been proposed as a remediation method for the large area of enriched soils around the refinery. Therefore, using the Ni hyperaccumulator Alyssum murale, we examined how soil Ni speciation influenced Ni availability and the subsequent mechanism of metal acquisition, translocation and storage. Using micro-XRF, micro-XAFS and micro-tomography we found that Ni is present throughout the leaf and stem tissues and co-localized with manganese at the base of the leaf trichomes. Fluorescence tomography verified this co-localization and revealed the enrichment of the dermal and vascular tissues. Absorption edge tomography of freshly removed leaves showed that the pattern of Ni compartmentalization was consistent throughout the entire leaf with an apparent concentration of Ni at the leaf tip. Micro-XAFS spectra from freshly harvested leaves and stems show that complexation with organic acids in the plant sap and leaf cell tissues are responsible for transport and storage of Ni. These results show that A. murale is capable of removing and concentrating Ni from these soils, however, the persistence of the relatively insoluble NiO particles may limit the effectiveness of this technique to fully remediate the site. Understanding how soil metal speciation affects plant metal availability and the mechanisms of metal accumulation and compartmentalization will help when making decisions as to how to remediate sites enriched with heavy metals.