Fungi counteract extracellular osmotic pressure by producing intracellular polyols to prevent loss of water. In yeast osmotic signaling involves a MAP-kinase pathway culminating at the STRE-binding transcription factors Msn2/4. We investigated the role of a putative STRE-binding orthologue of Trichoderma atroviride, Seb1, in osmotic stress signaling. T. atroviride, subjected to osmotic stress (10% glucose or glycerol, 1M KCl or NaCl), responds by raising its intracellular glycerol level. In contrast to Aspergillus nidulans, no erythritol is accumulated. Accumulation of glycerol levels under osmotic stress is strongly reduced in a seb1 deletion strain. To investigate glycerol biosynthesis in T. atroviride, the genes encoding glycerol dehydrogenase (gld1) and glycerol-3-phosphate dehydrogenase (gfd1) were cloned and characterized. Although both genes contain STRE-elements in their 5'-non-coding regions, only gld1 mRNA accumulates in response to osmotic stress, whereas expression of gfd1 remains at a constitutive level. In comparison to A. nidulans gld1 transcript levels in T. atroviride rise very slowly under conditions of salt stress. Deletion of seb1 results in a delayed accumulation of the gld1 transcript, but final levels match those in the wild-type whereas gfd1 transcript accumulation remains unaffected. Assays for glycerol dehydrogenase and glycerol-3-phosphate dehydrogenase enzymatic activities reveal an increase of the former--whereas the latter remains mainly unaffected--in the wild-type and the Deltaseb1 strain under different kinds of osmotic stress. The data suggest that Seb1 is only involved in, but not essential for osmotic stress response which is in contrast to the yeast orthologues Msn2/4.