Osmotic stress activates the expression of many plant genes through ABA-dependent as well as ABA-independent signaling pathways. We report here the characterization of a novel mutant of Arabidopsis thaliana, hos5-1, which exhibits increased expression of the osmotic stress responsive RD29A gene. The expression of several other stress genes are also enhanced by the hos5-1 mutation. The enhanced expression is specific to ABA and osmotic stress because low temperature regulation of these genes is not altered in the mutant. Genetic analysis indicated that hos5-1 is a recessive mutation in a single nuclear gene on chromosome III. Double mutant analysis of hos5-1 and the ABA-deficient aba1-1 as well as the ABA-insensitive abi1-1 mutant indicated that the osmotic stress hypersensitivity of hos5-1 is not affected by ABA deficiency or insensitivity. Furthermore, combined treatments of hos5-1 with ABA and osmotic stress had an additive effect on RD29A-LUC expression. These results suggest that the osmotic stress hypersensitivity in hos5-1 may be ABA-independent. The germination of hos5-1 seeds was more resistant to ABA. However, the hos5-1 mutation did not influence stomatal control and only slightly affected the regulation of growth and proline accumulation by ABA. The hos5-1 mutation reveals a negative regulator of osmotic stress-responsive gene expression shared by ABA-dependent and ABA-independent osmotic stress signaling pathways.