To evaluate the usefulness of determinations of telomerase activity for distinguishing malignant from benign mesothelial lesions, immunohistochemical (using a rabbit polyclonal antibody and the peroxidase method; n = 68) and in situ hybridization (using sense and antisense oligonucleotide probes; n = 46) studies were made on malignant mesotheliomas (epithelioid, 39; sarcomatoid, 18, including 2 of the desmoplastic type; and biphasic, 11) and 19 benign mesothelial lesions (benign mesothelial hyperplasia, 3; and reactive pleuritis, 16). In addition, biochemical studies of telomerase activity were made in 9 of the malignant mesotheliomas. Telomerase activity was detected histochemically in all but one of the malignant mesotheliomas, but only in one (pleuritis) of the benign lesions, in which it was present only in activated lymphocytes. Antisense hybridization signals indicated the presence of telomerase mRNA mainly in the cytoplasm of the malignant cells. Sense probes gave negative results. Biochemical determinations revealed a strong telomerase activity in the 9 malignant mesotheliomas examined. This study demonstrates the usefulness of immunohistochemical staining for the evaluation of mesotheliomas. The required immunostaining can be performed using paraffin sections of formalin-fixed tissues.