Ectomycorrhizal (ECM) fungi play important roles in fostering stress resistance of their host trees. In addition to nutrient benefits derived from colonization, ECM trees are also more resistant to toxic metals in soils. Aluminum (Al) in soil minerals is relatively stable under neutral conditions, but as pH decreases, the ionic, phytotoxic forms of Al increase in availability. We have investigated the Al resistance of Pisolithus tinctorius in vitro and measured the production of Al-chelating compounds and the distribution of Al between free and chelated forms in the mycosphere. Exudates were collected from P. tinctorius and analyzed for organic acids by ion chromatography. Media Al was analyzed by graphite furnace atomic absorption spectroscopy following strong cationic exchange fractionation. Al binding exudates were also separated by immobilized metal ion affinity chromatography (IMAC) and metal binding compounds characterized by high performance liquid chromatography. P. tinctorius was resistant to Al up to 400 然. Cultures produced abundant concentrations of organic acids, including tartrate (665 然), citrate (134 然), glycolate (1,236 然), formate (2,951 然) and acetate (457 然), which could chelate substantial quantities of Al. Al speciation analysis by fractionation indicated that P. tinctorius cultures chelated between 52 and 92% of free Al in the mycosphere. HPLC analysis of Al binding compounds isolated by IMAC indicated nine peaks were consistently present in four groups with retention times for each group ranging from 13-15 minutes, 16-18 minutes, 21-22 minutes, and 23-27 minutes. The last group contains the characteristic brown pigment produced by P. tinctorius. It appears that Al chelation and detoxification by P. tinctorius occurs through the action of a variety of fungal exudates.