Eucalyptus camaldulensis can detoxify Al by forming Al–citrate complexes, and this is achieved through citrate accumulation via suppression of the citrate decomposition pathway. •The citrate content in the roots was increased with Al treatments.•The activities of aconitase and NADP+-ICDH were decreased with Al treatments.•The activity of citrate synthase was not affected by Al.•Al–citrate complexes were a major chemical form in the cell sap of root tips. Eucalyptus (Eucalyptus camaldulensis) has relatively high resistance to aluminum (Al) toxicity than the various herbaceous plants and model plant species. To investigate Al-tolerance mechanism, the metabolism of organic acids and the chemical forms of Al in the target site (root tips) in Eucalyptus was investigated. To do this, 2-year old rooted cuttings of E. camaldulensis were cultivated in half-strength Hoagland solution (pH 4.0) containing Al (0, 0.25, 0.5, 1.0, 2.5 and 5.0mM) salts for 5weeks; growth was not affected at concentrations up to 2.5mM even with Al concentration reaching 6000μgg−1 DW. In roots, the citrate content also increased with increasing Al application. Concurrently, the activities of aconitase and NADP+-isocitrate dehydrogenase, which catalyze the decomposition of citrate, decreased. On the other hand, the activity of citrate synthase was not affected at concentrations up to 2.5mM Al. 27Al-NMR spectroscopic analyses were carried out where it was found that Al–citrate complexes were a major chemical form present in cell sap of root tips. These findings suggested that E. camaldulensis detoxifies Al by forming Al–citrate complexes, and that this is achieved through Al-induced citrate accumulation in root tips via suppression of the citrate decomposition pathway.