Abstract:The haploid and diploid calli were induced respectively from anther and immature embroy cultures in barley cultivar ‘Zhaoshu No.3’, which is susceptible to acid-aluminum. In order to establish the selection conditions, these calli were cultured on the media with different pH values and aluminum concentrations to examine response to acid-aluminum. It was showed that pH 6–8 was optimal for the growth of calli and pH values under 5 or over 10 inhibited the growth of calli significantly and the selection medium was at pH 4.5 and 10 ppm Al+3. The callus lines toleranted to 10–20 ppm A1+3 have been selected from calli treated with 5000 red γ-ray radiation or no treatment. In the conditions of pH 4.5 and 10 ppm Al+3, the cells of original type were irregularly ellipsoide, dimly fluorescent and low respiratory intensity and didn’t grow, while the cells of Al-tolerant callus line were spherical, brilliant fluorescent and high respiratory intensity and continued to grow. lsozymes of peroxidase in Al-tolerant variant was somewhat different from that of original type. No plant was regenerated from Al-tolerant haploid calli, but 5 regenerated plants were obtained from diploid calli in selected subculture. Two of them survived were examined to be tolerant to pH 4.5, 10–20 ppm A1+3 and were able to grow in acid-aluminumrodish soil. Finally, the application of in vitro culture and selection for genetic improvement of Al-tolerance in barley was discussed briefly on the basis of the results obtained in the present study.