CD44 isoforms belong to a family of cell adhesion molecules expressed on the cell surface of many tumor cells during human breast cancer progression. In this study we have analyzed the expression of CD44v3-containing isoforms [containing heparan sulfate addition sites for growth factor binding] in primary breast tumors, axillary nodal metastases and normal breast tissue. Using reverse transcriptase-polymerase chain reaction (RT-PCR) followed by Southern blot, cloning, nucleotide sequencing and RT-in situ-PCR analyses, we have found that at least two CD44v3-containing isoforms, including one new species of CD44v2,∆v3-10 (deltav3 defined as a v3 exon lacking the first 24 base pairs) and another previously reported CD44v3,8-10 are preferentially expressed in human primary breast tumor and axillary nodal metastases but not in normal breast tissues. These finding suggest that these CD44v3-containing isoforms are closely associated with breast cancer metastasis. Furthermore, we have established a stable transfection of CD44v2,∆v3-10 cDNA into non-metastatic human breast tumor cells (MCF-7) which contain endogenous CD44E isoform. Our results indicate that expression of CD44v2,∆v3-10 in MCF-7 cells promotes tumor cells to undergo rapid cell growth and active cell migration. Treatments of MCF-7 transfectants expressing CD44v2,∆v3- 10 with various agents such as anti-CD44v₃ antibody, cytochalasin D (a microfilament disrupting agent known to prevent actin polymerization) and W-7 (a calmodulin antagonist) but not colchicine (a microtubule inhibitor), cause a significant inhibition of tumor cell migration. These findings suggest that CD44v2,∆v3-10 (related to human metastatic breast cancers) and associated microfilament components play an important role in the regulation of breast tumor cell behaviors required for the progression of human breast carcinomas.