This study reports the establishment of three major subtypes of human mesothelioma cells in tissue culture, i.e. the epithelioid, sarcomatoid and biphasic forms, and compares their phenotypic and biological characteristics. Primary cells isolated from biopsies or pleural exudates were subcultured for over 50 passages. We evaluated immunoreactivity using various mesothelial markers related to histological patterns of these cell lines. For epithelioid cells, calretinin and cytokeratin were found to be useful and easily interpretable markers as for control mesothelial cells. The biphasic form was only partially positive and the sarcomatoid type negative. Vimentin was expressed by all cell lines. BerEP4, a specific marker for adenocarcinoma, was negative. Interestingly, while the macrophage marker CD14 was negative, immunoreactivity for a mature macrophage marker (CD68) was expressed by all cell types, suggesting that this marker might constitute an additional tool useful in the differential diagnosis of mesothelioma. At the ultrastructural level, a cell surface rich in microvilli confirmed their mesothelial origin. PCR analysis revealed that none of the cell lines contained SV40 DNA. Karyotypic analyses showed more complex abnormalities in the epithelioid subtype than in the sarcomatoid form. These cell lines may be useful in the study of cellular, molecular and genetic aspects of the disease.