In the present study, we have employed a unique breast cancer cell line (Met-1, which was derived from a high metastatic potential tumor in transgenic mice expressing polyomavirus middle T oncogene) to study the role of CD44 variant isoform(s) in the regulation of metastatic breast tumor cell behavior. The results of reverse transcriptase-polymerase chain reaction, Southern blot, nucleotide sequencing, immunoprecipitation, and immunoblot analyses indicated that these cells express a major CD44 isoform (molecular weight É 260 kDa) containing a v3,8-10 exon insertion (designated as CD44v 3,8-10 ). In addition, we have determined that CD44v 3,8-10 binds specifically to the cytoskeletal proteins such as ankyrin. Biochemical analyses, using competition binding assays and a synthetic peptide identical to NGGNGTVEDRKPSEL (a sequence located between aa480 and aa494 of CD44v 3,8-10 ) indicate that this 15-amino acid peptide binds specifically to the cytoskeletal protein ankyrin (but not to fodrin or spectrin). This peptide competes effectively for ankyrin binding to CD44v 3,8-10 . Therefore, we believe that the sequence 480 NGGNGTVEDRKPSE 494 L, located at the cytoplasmic domain of CD44v 3,8-10 , is required for the ankyrin binding. We have also detected that CD44v 3,8-10 -containing Met-1 cells are capable of forming membrane spikes or ''invadopodia'' structures and undergo active migration processes. Treatments of Met-1 cells with certain agents including anti-CD44v 3 antibody, cytochalasin D (a microfilament inhibitor), and W-7 (a calmodulin antagonist), but not colchicine (a microtubule disrupting agent) effectively inhibit ''invadopodia'' formation and subsequent tumor cell migration. Further analyses using zymography assays and double immunofluorescence staining indicated that CD44v 3,8-10 is closely associated with the active form of matrix metalloproteinase, MMP-9, in a complex within ''invadopodia'' structures. These findings suggest that CD44v 3,8-10 plays an important role in linking ankyrin to the membrane-associated actomyosin contractile system required for ''invadopodia'' formation (coupled with matrix degradation activities) and tumor cell migration during breast cancer progression.