Subcellular fractionation experiments with mouse hepatocytes, combined with sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis (PAGE)–immunoblot analysis using antibodies against two different tail regions of mouse myosin-X demonstrated a 240 kDa molecular mass to be associated with the plasma membrane-rich P2 fraction. The basolateral plasma membrane fraction, but not the brush border fraction, isolated from renal cortices also contained the 240 kDa form of myosin-X. In an attempt to assess relative contributions of possible functional domains in the tail of myosin-X to localization and function, cDNA corresponding to all three pleckstrin homology (PH) domains and different regions (PH1, 2 and 3, and the two subdomains of PH1: PHS1 and PHS2), as well as the myosin tail homology 4 domain (MyTH4) and the band4.1/ezrin/radixin/moesin-like domain (FERM) were separately inserted into the pEGFP vector and expressed in cultured COS-1 cells. As a result, two distinct regions responsible for localization were identified with regard to PH: one covers all three forms that tends to localize to regions of dynamic actin, such as membrane ruffles, lamellipodia and thick cortical actin bundles at the sites of cell–cell adhesion in a Rac- and Cdc42-dependent manner. The other covers PHS1 and PH2 that localizes to filopodia, filopodial puncta and the sites of intercellular adhesion in a Cdc42-dependent manner. Expression of green fluorescent protein (GFP)-MyTH4 fusion protein resulted in formation of phalloidin-positive granules, while GFP-FERM affected the actin cytoskeletal system in a distinctly different way. Taken altogether, the results lend support to the view that myosin-X is involved in cell–cell adhesion-associated signaling-linked membrane and/or cytoskeleton reorganization.