A complementary DNA (cDNA) library was constructed from a human malignant mesothelioma (MM) cell line and a cDNA fragment encoding for a cytoplasmic mesothelial protein recognized by the polyclonal antibody AMAD-1 was then cloned and expressed in Escherichia coli. The purified recombinant protein was used to raise a novel antibody, named AMAD-2, in rabbits. This antibody reacted with normal mesothelium and most MM (15 of 17) on paraffin sections and featured a cytoplasmic labeling. Conversely, AMAD-2 immunostaining of normal and tumor tissues from body sites other than serosal membranes was limited with respect to the proportion of positive specimens and usually less conspicuous than in MM. AMAD-2 immunoreactivity was subsequently compared with staining for HBME-1, another newly marketed antimesothelial monoclonal antibody, concerning the ability to distinguish pleural MM from metastatic pleural tumors of epithelial type. A granular cytoplasmic immunoreactivity for AMAD-2 was present in 50% or more of tumor cells in all 84 MM, regardless of histological type, but also in 3 (7%) of 42 pleural metastases, albeit only focally. HBME-1 was shown in 63 of 66 epithelial MM and in the epithelial component of all 8 mixed MM, with a prevailingly membranous pattern, usually homogeneous and strong, whereas none of the 10 sarcomatous MM was positive. HBME-1 was also expressed in 6 (14%) of 42 pleural metastases in a cytoplasmic or membranous pattern. Compared with HBME-1, AMAD-2 showed a higher degree of specificity and sensitivity for MM. AMAD-2 still proved to be superior to HBME-1, also when sarcomatoid MM were excluded from the assessment. This finding supports the view that AMAD-2 is an antibody highly, although not entirely, specific for the mesothelial lineage, whereas HBME-1 is probably a cell marker more closely related to the epithelial differentiation of MM. Therefore, AMAD-2 is preferable as a positive tissue marker to be incorporated in the optimal immunohistochemical panel for the diagnosis of MM.