Pleomorphic adenoma is the most common epithelial tumor in the salivary glands. This tumor frequently exhibits "mesenchyme"-like components, including myxoid or chondroid areas. Recently, using immunohistochemical techniques, we reported that cartilage-specific matrix protein, chondromodulin-I (ChM-I), was deposited on the inter-territorial matrix of the chondroid area in salivary pleomorphic adenomas and that ChM-I, which is also a strong angio-inhibitory factor, plays an important role in the avascular nature of the chondroid area and the chondroid formation in this type of tumor. To elucidate which cells express ChM-I mRNA in pleomorphic adenomas, we examined the expression and localization of ChM-I mRNA in this type of tumor using an in situ hybridization technique. Immunoreactivity for ChM-I was observed in the inter-territorial matrix of the chondroid area, especially around the lacunae, and in the cytoplasm of neoplastic myoepithelial cells of the myxoid element of pleomorphic adenomas. On in situ hybridization analysis, strong signals for ChM-I mRNA were detected in the cytoplasm of the lacuna cells of the chondroid element, and moderate to marked signals were observed in the cytoplasm of the neoplastic myoepithelial cells of the myxoid element. Signals for ChM-I mRNA were also seen in the cytoplasm of the spindle-shaped neoplastic myoepithelial cells in the transitional areas between the myxoid and chondroid elements of this tumor. Signals for ChM-I mRNA were not seen in the inner ductal cells or the fibrous element. These findings indicate that lacuna cells and neoplastic myoepithelial cells express ChM-I mRNA and that mature ChM-I, which lacuna cells and neoplastic myoepithelial cells translate, is deposited in the chondroid matrix of pleomorphic adenomas. In conclusion, lacuna cells and neoplastic myoepithelial cells express ChM-I mRNA ectopically in pleomorphic adenoma, and this plays an important role in chondroid formation and hypovascularity in this type of tumor.