Barley (Hordeum vulgare L.) is classified as an aluminum (Al) sensitive species, and improvement of its Al tolerance is essential for barley grown in acid soils. Al-stimulated efflux of organic acids has been observed in several plant species, and the efflux is correlated well with Al tolerance of these species. However, limited information on mechanisms of Al tolerance is available for barley. In the present study, 12 Al-tolerant cell lines of barley were obtained through mutagenesis of ethyl methane sulfonate (EMS), NaN 3 and gamma ray. Four Al-tolerant mutation cell lines were selected and characterized for their mechanisms of Al tolerance. After the four mutant cell lines (M 3 , M 1 0 , M 1 1 and M 2 5 ) were treated with 30 μM Al for 24 h, their relative growth in a modified MS medium was at least 26% higher than the parental line (PL) at day 10. Al accumulation in cells of the three mutant cell lines (M 3 , M 1 0 and M 2 5 ) was significantly lower than that in the parental cell line. Organic acid analysis revealed that Al-stimulated release of citrate and/or malate was enhanced in M 3 , M 1 0 and M 2 5 , which is well correlated with the improved Al tolerance in these three mutant cell lines. This indicates that Al-exclusion mechanism by organic acids is operating in barley cell lines like other grass species. However, there were no significant differences in Al accumulation of cells and the concentrations of internal and external citrate or malate between M 1 1 and the PL, which suggests that other mechanisms of Al tolerance other than Al-stimulated exudation of organic acids may exist in barley. In addition, Al-stimulated exudation of citrate or malate resulted in decrease of intracellular concentrations of citrate or malate in all of the four mutation cell lines. This shows that Al-stimulated exudation of organic acids is independent of organic acid synthesis. Therefore, it is essential to enhance Al-stimulated efflux of organic acids for improvement of Al tolerance in barley.