Knowledge of the intracellular distribution of Cd in leaves is necessary in order to understand the mechanisms of hyperaccumulation in Thlaspi caerulescens. Ganges and Prayon, two ecotypes accumulating Cd to different levels, were grown in nutrient medium containing varying concentrations (0, 5, 10, 50, and 100 microM) of Cd. Several different approaches were combined in this study to (i) validate the results obtained by a specific method and (ii) establish the link between observations and measurements performed at different scales. In both ecotypes, Cd, localized by autoradiography, was found mainly at the edges of the leaves, but also in points of higher concentration spread over the whole limb surface. This localization was clearly correlated with the necrotic spots observed on Prayon leaves. Scanning electron microscopy coupled with energy dispersive X-ray microanalysis (cryo-SEM-EDXMA) and tissue fractionation (apoplasm, cell walls, mesophyll protoplasts, and lower epidermis) showed that Cd had similar patterns of distribution in leaf cells of both ecotypes. Cadmium was found both inside the cells and in the cell walls, mainly in the large epidermal cells but also in small epidermal cells. All the methods used agreed well and the results indicated that metal storage in the plants studied involves more than one compartment and that Cd is stored principally in the less metabolically active parts of leaf cells.