Histone deacetylases (HDACs) were originally identified as nuclear enzymes involved in gene transcription regulation. Until recently, it was thought that their activity was restricted within the nucleus, with histones as unique substrates. The demonstration that specific HDACs deacetylate nonhistone proteins, such as p53 and alpha-tubulin, broadened the field of activity of these enzymes. HDAC8, a class I HDAC, is considered to be ubiquitously expressed, as suggested by results of Northern blots performed on tissue RNA extracts, and transfection experiments using various cell lines have indicated that this enzyme may display a prominent nuclear localization. Using immunohistochemistry, we unexpectedly found that, in normal human tissues, HDAC8 is exclusively expressed by cells showing smooth muscle differentiation, including visceral and vascular smooth muscle cells, myoepithelial cells, and myofibroblasts, and is mainly detected in their cytosol. These findings were confirmed in vitro by nucleo-cytoplasmic fractionation and immunoblot experiments performed on human primary smooth muscle cells, and by the cytosolic detection of epitope-tagged HDAC8 overexpressed in fibroblasts. Immunocytochemistry strongly suggested a cytoskeleton-like distribution of the enzyme. Further double-immunofluorescence staining experiments coupled with confocal microscopy analysis showed that epitope-tagged HDAC8 overexpressed in murine fibroblasts formed cytoplasmic stress fiber-like structures that co-localized with the smooth muscle cytoskeleton protein smooth muscle alpha-actin. Our works represent the first demonstration of the restricted expression of a class I HDAC to a specific cell type and indicate that HDAC8, besides being a novel marker of smooth muscle differentiation, may play a role in the biology of these contractile cells.