Although short-chain fatty acid (SCFA)-induced heat shock protein 25 (Hsp25) is associated with increased cellular resistance to injury, withdrawal of lumenal butyrate in vivo is associated with intestinal epithelial injury and apoptosis. Recognizing that SCFA-dependent post-translational modification of Hsp25 may involve altered Hsp25 phosphorylation, we hypothesized that butyrate regulates Hsp25 phosphorylation and secondarily affects cellular responses to apoptosisinducing agents. IEC-18 cells were treated with butyrate, propionate or the histone deacetylase inhibitor trichostatin A for 6-24 hrs. Immunolocalization of Hsp25 was examined by confocal laser microscopy. Hsp25 phosphorylation was characterized using 2-D iso-electric focusing gel electrophoresis. Hsp25 accumulation in cytoskeletal- and mitochondrial-enriched fractions was examined by immunoblotting. The activation of p38 MAP kinase was determined using phospho-specific antibodies and MAPKAPK 2 kinase assays. The effects of SCFA on apoptosis were studied by ELISA detection of cleaved DNA and using antibodies recognizing cleaved caspase 3. Five-millimolar butyrate induced no significant injury to IEC-18 cells. Hsp25 did not accumulate in Triton X-100 insoluble cytoskeletal fractions with butyrate treatment but did localize to mitochondria in a p38 MAP kinase-dependent manner. Hsp25 phosphorylation was induced by butyrate, propionate and trichostatin A. Butyrate-mediated changes in Hsp25 phosphorylation coincide with the activation of the p38 MAP kinase and MAPKAPK 2. Butyrate, propionate and low-dose trichostatin A confer significant protection from camptothecin-induced apoptosis which was not reversed by the p38 inhibitor SB203580. We conclude that butyrate-mediated phosphorylation of Hsp25 is associated with significant resistance to apoptosis which appears to be independent of p38-mediated targeting of Hsp25 to mitochondria.