This is the first report about human tenascin-W, the fourth and final member of the extracellular matrix protein family of tenascins. Sixty-three human breast tumor extracts were analyzed by Western blotting for the presence of tenascin-W and compared with tenascin-C, an established marker of tumor stroma. Interestingly, we found tenascin-W expression in the majority of the tumor tissues, but no detectable expression in the normal mammary parenchyma. Eighty-one percent of the breast tumor samples were tenascin-W positive and 86% showed expression of tenascin-C. However, tenascin-W and tenascin-C amounts varied greatly between tumors and some contained either tenascin-W or tenascin-C exclusively, indicating independent mechanisms regulating their expression. Although there was no difference between high- or low-grade tumors with respect to the presence of tenascin-C, tenascin-W was more prominent in low-grade tumors. For 42 of the breast cancer tissues, a frozen tumor microarray was available to confirm the Western blot data by immunohistochemistry. Similar to tenascin-C, tenascin-W was detected in the tumor stroma. Fibroblasts adhered to tenascin-W in a beta(1) integrin-dependent manner and spread with a distinctive morphology under conditions where they remained round on tenascin-C. CHOB2 cells expressing alpha(v)beta(1) or alpha4beta(1) integrins were able to spread on tenascin-W. Furthermore, addition of tenascin-W to the culture medium increased migration of breast cancer cells toward a fibronectin substratum in vitro. These data imply that tenascin-W expression in the activated tumor stroma facilitates tumorigenesis by supporting the migratory behavior of breast cancer cells.