Heat stress alters epithelial barrier function, and heat stress preconditioning protects epithelial function from injury. Hsp27 is a small stress protein that has previously been shown to modulate actin assembly. Thus, by regulating actin filaments associated with cell junctions, hsp27 could alter epithelial function. To begin to address this hypothesis, the regulation and distribution of a human hsp27-green fluorescence fusion protein ((EGFP)hHsp27) that is expressed in cultured renal epithelial cells was assessed. (EGFP)hHsp27, like the endogenous hsp27, associated with the cytoskeleton in heat-stressed and chemically ATP-depleted cells, and both proteins were regulated similarly. Confocal microscopy of intact and detergent-lysed cells revealed novel distribution patterns in which (EGFP)hHsp27 associated with basolateral, but not apical, cell borders in injured cells. Double labeling studies revealed (EGFP)hHsp27 and actin filament colocalization in ATP-depleted cells. However, during heat shock, granules of (EGFP)hHsp27 were found at sites of cell-cell contact and in the cell body, but colocalization with actin was not apparent. Thus, heat stress and ATP depletion induce distinct patterns of hsp27 redistribution in epithelial cells, and sites of cell-cell and cell-substrate attachment are unique in their ability to recruit hsp27 during injury. The association of (EGFP)hHsp27 with basolateral cell boundaries supports a potential role for hsp27 in protection or regulation of epithelial cell-cell and cell-substrate attachments.