Myofibroblasts and extracellular matrix protein tenascin-C (Tn-C) are known to be implicated in cancer progression in human cancer. In feline mammary tumors that are a suitable model for human breast cancer, however, little is known about stromal myofibroblasts and no information is available on the expression of Tn-C. Feline samples of normal mammary glands and proliferating mammary lesions were routinely processed and serial sections were cut and immunostained with anti-α-smooth muscle actin (α-SMA) or Tn-C antibody. Myofibroblasts were not included in the stroma of 90% (9/10) of normal mammary gland tissues, 92% (12/13) of adenosis, and 63% (5/8) of simple adenomas. On the other hand, all 40 simple carcinomas contained stromal myofibroblasts to a varied extent. Tn-C expression was detected in the stroma of 92% (37/40) of carcinomas, and its global distribution almost coincided with that of myofibroblasts. In addition, Tn-C immunoreactivity was occasionally observed in the basement membrane zone around ducts in some cases of normal mammary glands and benign lesions, but barely observed in the stroma. These results suggest that stromal myofibroblasts may be a major cellular source of Tn-C and be involved in malignant progression of feline mammary tumor.