Tropoelastin protein monomers assemble to form elastin.  Cellular integrin a_Vb₃ binds RKRK at the carboxy-terminal tail of tropoelastin. We probed cell interactions with tropoelastin by deleting the RKRK sequence in order to identify other cell binding interactions within tropoelastin. We found a novel human dermal fibroblast attachment and spreading site on tropoelastin that is located centrally in the molecule. Inhibition studies demonstrated that this cell adhesion was not mediated by either elastin binding protein or glycosaminoglycans. Cell interactions were divalent cation-dependent, indicating integrin dependence. Function blocking monoclonal antibodies revealed that a_V- integrin(s) and integrin a_Vb₅ specifically were critical for cell adhesion to this part of tropoelastin. These data reveal a common a_V integrin binding theme for tropoelastin: a_Vb₃ at the C-terminus and a_Vb₅ at the central region of tropoelastin.Each a_V region contributes to fibroblast attachment and spreading but they differ in their effects on cytoskeletal assembly.