Myoepithelial cells of salivary glands have a complex cytoskeletal immunophenotype. To elaborate the smooth muscle phenotype of salivary gland myoepithelium and to assess its contribution to the histogenesis of pleomorphic adenomas, we evaluated the immunohistochemical expression of three novel monoclonal antibodies (MAbs) to alpha smooth muscle actin (alpha-SMA), smooth muscle myosin heavy chains (SMMH), and calponin in formalin-fixed tissues of 65 pleomorphic adenomas (51 contained surrounding normal salivary gland as well). Different cell types within the pleomorphic adenomas were classified as inner tubular epithelial cells, myoepithelium-like cells (juxtatubular, cuboidal, and spindle), modified myoepithelium (myxoid, chondroid, hyaline), and transformed myoepithelium (solid epithelioid, squamous, basaloid-cribriform). Periacinar and periductal myoepithelial cells of all of the 51 normal salivary glands were diffusely stained by all of the 3 MAbs, whereas all of the acinar/ductal epithelial cells were entirely negative. Of 65 pleomorphic adenomas, 61 (94%) reacted to all of the 3 MAbs. None of the smooth muscle markers stained the inner-tubular epithelial cells. Both alpha-SMA and SMMH were essentially limited to the myoepithelium-like cells, whereas modified and transformed myoepithelia lacked these myofilaments. Calponin was found in 64 (98%) of the tumors, reacting to almost all of the myoepithelium-like cells, to 60% of the modified myoepithelium, and to 30% of the transformed myoepithelium. We found the expression of these smooth muscle-specific proteins in the neoplastic myoepithelium to be associated with morphologic differentiation. Alpha-SMA and SMMH are only expressed in better differentiated neoplastic myoepithelium. Calponin is the most sensitive marker of neoplastic myoepithelium, and its identification in different cell types of pleomorphic adenomas denotes a major histogenetic role of myoepithelial cells.