Plant species differ in response to high available manganese (Mn), but the mechanisms of sensitivity and tolerance are poorly understood. In solution culture, {greater than or equal to} 30 µM Mn decreased the growth of soybean (Glycine max) but white lupin (Lupinus albus), narrow-leafed lupin (L. angustifolius), and sunflower (Helianthus annuus) grew well at 100 µM Mn. Differences in species' tolerance to high Mn could not be explained simply by differences in root, stem, or leaf Mn status, being 8.6, 17.1, 6.8, and 9.5 mmol kg-1 leaf fresh mass at 100 µM Mn. Furthermore, X-ray absorption near edge structure (XANES) analyses identified the predominance of Mn(II), bound mostly to malate or citrate, in roots and stems of all four species. Rather, differences in tolerance were due to variations in Mn distribution and speciation within leaves. In Mn-sensitive soybean, in situ analysis of fresh leaves using X-ray fluorescence microscopy (µ-XRF) combined with XANES showed high Mn in the veins and manganite [Mn(III)] accumulated in necrotic lesions apparently through low Mn sequestration in vacuoles or other vesicles. In the two lupin species, most Mn accumulated in vacuoles as either soluble Mn(II) malate or citrate. In sunflower, Mn was sequestered as manganite at the base of non-glandular trichomes. Hence, tolerance to high Mn was ascribed to effective sinks for Mn in leaves - as Mn(II) within vacuoles or through oxidation of Mn(II) to Mn(III) in trichomes. These two mechanisms prevented Mn accumulation in the cytoplasm and apoplast, thereby ensuring tolerance to high Mn in the root environment.