A hydroponic experiment was carried out to study the alleviating effects of exogenous betaine on drought stress in barley seedlings (Hordeum vulgare L.) cv. ZJU9. Results showed that foliar applications of 500 mu M betaine ameliorated drought-induced reductions in plant height, root length and root dry weight and improved chlorophyll contents and photosynthesis parameters, compared with seedlings given a drought alone treatment. In addition, exogenous betaine significantly increased root and leaf SOD, POD, APX and root CAT activities (cf. 15.5%/19.8%, 13.5%/7.9%, 10.0%/31.9%, 10.45%/11.3% higher than drought alone treatment), markedly reduced Na+ concentrations and elevated the K+/Na+ ratio in both shoots and roots by 39.0, 38.7, 50.7 and 42.6%. Also, betaine ameliorated drought-induced damage to leaf and root ultrastructure, effectively improving the stability and integrity of the nuclear membrane of root meristem cells and increased cell viability in roots tips. The results suggest that betaine alleviates drought-induced oxidative damage in plants via stimulating antioxidative enzyme activities and K uptake, and helps to maintain stability and integrity of the sub-cellular structure under drought stress.