Among the many sensors and transducers involved in cell volume regulation, the cytoskeleton plays an important role activating a series of cascading events that bear the cell to the original hydration state. The aims of the present work are the characterization of the F-actin cytoskeleton in rat colonocytes by confocal microscopy and the evaluation of possible F-actin rearrangements following exposure of the tissue to hypotonic and hypertonic stress. The study was carried out on paraformaldehyde fixed colon criosections stained with rhodamine-phalloidin. In rat colonocytes actin is mainly localized along the peripheral part of the cell defining the cortical cytoskeleton, with greater rhodamin staining in the brush-border region. In hypertonic stress condition the cortical cytoskeleton showed a significant constriction after 5 minutes of stress exposure, as indicated by the decrease of cytoskeleton perimeter, followed by a Regulatory Volume Increase (RVI) response after 30 minutes of stress exposure. In hypotonic conditions the cortical cytoskeleton showed an expansion, followed by a Regulatory Volume Decrease (RVD) response which reported the cells to the initial size. The results suggest that the changes undergone by the cytoskeleton in the course of the osmotic shrinkage and swelling represents a "sensor" of the cell volume changes. Therefore, F-actin skeleton, associated with ion membrane transporters, would play a crucial role in the RVI and RVD activation in rat colon.