Dynamin is a large mechanochemical GTPase that has been implicated in vesicle formation in multiple cellular compartments. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. To identify potential intracellular proteins that interact with the PH domain of dynamin II, we carried out a yeast two-hybrid screen in which the PH domain of dynamin II was used as bait. The cell surface heparan sulfate proteoglycan syndecan-4 that acts in conjunction with integrins to promote the formation of actin stress fibers and focal adhesions was isolated as a binding partner for the PH domain of dynamin II. In vitro binding assays, immunoprecipitation, and confocal microscopy analysis confirmed the association of dynamin II with syndecan-4. Most dramatic finding of our study is that the cytoplasmic distribution of dynamin II and syndecan-4 changes in fibroblasts that have been stimulated to form the focal adhesions and stress fibers with LPA. In quiescent cells, dynamin II is evenly distributed in the cytoplasm and colocalizes with syndecan-4 near the nucleus. Upon treatment with LPA to induce focal adhesions and stress-fiber formation, dynamin II becomes markedly associated with syndecan-4 at focal adhesion sites. We further established the colocalization of syndecan-4 and dynamin with paxillin and actin as marker proteins for focal adhesions and stress fibers, respectively. All of these results suggest that the interaction between dynamin II and syndecan-4 is important in mediating focal adhesion and stress-fiber formation.