Knowledge of the mechanistic basis of differential aluminum (Al) tolerance depends, in part, on an improved ability to quantify Al located in the apoplastic and symplastic compartments of the root apex. Using root tips excised from seedlings of an Al-tolerant wheat cultivar (Triticum aestivum L. cv Yecora Rojo) grown in Al solutions for 2 d, we established an operationally defined apoplastic Al fraction determined with six sequential 30-min washes using 5 mm CaCl(2) (pH 4.3). Soluble symplastic Al was eluted by freezing root tips to rupture cell membranes and performing four additional 30-min CaCl(2) washes, and a residual fraction was determined via digestion of root tips with HNO(3). The three fractions were then determined in Yecora Rojo and a sensitive wheat cultivar (Tyler) grown at 18, 55, or 140 mum total solution Al (Al(T)). When grown at equal Al(T), Tyler contained more Al than Yecora Rojo in all fractions, but both total Al and fractional distribution were similar in the two cultivars grown at Al(T) levels effecting a 50% reduction in root growth. Residual Al was consistently 50 to 70% of the total, and its location was elucidated by staining root tips with the fluorophore morin and examining them using fluorescence and confocal laser scanning microscopy. Wall-associated Al was only observed in tips prior to any washing, and the residual fraction was manifested as distinct staining of the cytoplasm and nucleus but not of the apoplastic space. Accordingly, the residual fraction was allocated to the symplastic compartment for both cultivars, and recalculated apoplastic Al was consistently approximately 30 to 40% of the total. Distributions of Al in the two cultivars did not support a symplastic detoxification hypothesis, but the role of cytoplasmic exclusion remains unsettled.