The presence of myofibroblasts in the stroma of Phyllodes tumor of the breast has been documented in single cases by means of electron microscopy and immunohistochemistry. In this study, we re-evaluate the immunophenotype exhibited by 28 Phyllodes tumors (14 benign, 6 borderline and 8 malignant) in order to state the relevance of myofibroblasts as active stromal elements in these tumors. Vimentin showed positive immunostaining in the stroma of all the cases (100%), muscle-specific actin in 21 (75%), and desmin in 7 (25%). By contrast, stromal cells were uniformly negative for S-100 protein, keratins (AE1/AE3 and CAM 5.2), and epithelial membrane antigen (EMA). The epithelial component showed the typical two-layered arrangement of ducts in every case. Myoepithelial (outer) cells immunostained with muscle-specific actin and S-100 protein, and in some cases with EMA and keratins, whereas the epithelial (inner) layer did so with keratins and EMA. To conclude, we have found a prominent myofibroblastic differentiation in stromal cells of many of such tumors. Our results also prove that no differences in intensity or distribution of such actin-positive stromal cells are seen between benign, borderline or malignant counterparts.