Abstract: Objective To establish an method for detecting human telomerase reverse transcriptase (hTERT) mRNA of cellsin pleural fluid by in situ hybridization (ISH), and to evaluate preliminarily the efficacy of this new method in recognizing neoplastic cells in the pleural fluid. Methods Fresh pleural fluid specimens were collected in 23 patients with pleural effusions and cell smears were prepared and after pretreatment, ISH between hTERT mRNA of the cells and digoxin-labelled oligonucleotide probes was performed. The signals of hTERT mRNA were detected by the sequential treatment with antigen, antibody, enzyme, and staining. With strict negative and positive controls, the experimental study was performed independent of clinical diagnosis and treatment. Results Positive results were seen in 9 cases of malignant pleural effusions, where dark staining of the cytoplasm and a few stained spots in the cell nuclei could be observed microscopically irrespective of the cytomorphological findings. Seven of the 9 cases had positive results of cytomorphological examination, in which neoplasmic cell with obvious morphological abnormalities were also spotted. In the other 2 cases where cytomorphological findings were negative but pleural biopsy confirmed the diagnosis of malignant pleural effusions, cytoplasm dark staining with stained spots in the cell nuclei were seen under microscope in spite of the absence of obvious abnormality in cell morphology. In contrast, the 14 cases with benign pleural effusions all had negative results, in which neither staining of the cells was identified microscopically, nor was evidently abnormal cell morphology. Conclusions As a new method of clinical cytopathology, detection of hTERT mRNA of cells in the pleural fluid by ISH may provide a new means for cytomorphological examination for differential diagnosis between benign and malignant pleural effusions and for classification of the identified tumor cells.