Changes in actin cytoskeleton in the C6 rat glial cell line were studied during decrease or increase (abrupt or gradual) of extracellular osmolality. Actin cytoskeleton was visualized by confocal microscopy after FITC-phalloidin labeling. G-actin, Triton-soluble F-actin and Triton-insoluble F-actin subfractions were determined by gel electrophoresis and scanning, and by DNase I inhibition assays. In control conditions C6 glial cells exhibited well-defined stress fibers and a relatively smooth cortical network. Extracellular anisosmotic changes induced a rapid actin cytoskeletal reorganization, which further progressed and was not reversed upon cell volume recovery. Hypotonic shock caused membrane ruffling and a shift towards polymerized actin, whereas hypertonicity (abrupt or gradual) led to a distinct morphological appearance of abundant short actin microfilaments with, however, no detectable alteration in actin subfractions. When anisosmotic cell volume regulation was prevented, cytoskeleton reorganization depended on the osmotic change and the experimental protocol, but was not related to the absence of volume readjustment. Therefore, although involvement of cytoskeletal alterations in transduction of volume regulatory responses cannot be excluded, it is likely that the observed changes in actin cytoskeleton in C6 glial cells are linked with, but do not initiate, cell volume regulatory processes.