Linker histone protein variants are expressed in different tissues, at various developmental stages or induced by specific environmental conditions in many plant species. In most cases, the function of these proteins remains unknown. In the work presented here an antisense strategy has been used to study the function of the drought-induced linker histone, H1-S of tomato. Three independent H1-S antisense tomato mutants, selected for their inability to accumulate H1-S in response to water stress, were studied. These mutants have been characterized at the physiological and morphological levels. Histone H1-S antisense transgenic plants developed normally indicating that H1-S does not play an important role in the basal functions of tomato development. No differences were detected in chromatin organization, excluding a structural role for H1-S in chromatin organization. However, differences between the wild-type and antisense plants were observed in leaf anatomy and physiological activities. This analysis indicates that H1-S has more than one function, at different times, in controlling plant water status, highlighting the complexity of the water stress response.