As was reported that human mammaglobin (hMAM) may be expressed in malignant pleural effusions (PEs), we investigated the relevance of hMAM reverse-transcriptase polymerase chain reaction (RT-PCR) for their diagnosis and determination of primary origin. Two hundred and twenty-eight malignant (132 male, 96 female) and 185 benign (132 male, 53 female) PEs were investigated. Statistical analyses evaluated the diagnostic performance parameters in all PEs and in cytologically negative malignant PEs, the association between hMAM and benign or malignant status by the direct index of correlation [diagnostic odds ratio (DOR)], chi test, and P value (P). In addition, the discriminative diagnostic power of hMAM expression, independently in breast cancer, lung cancer (LC), malignant mesothelioma (MM), and other cancers was evaluated. In the entire patient population, hMAM was detected in 45.6% and 5.4% of malignant and benign PEs, respectively, in the male group in 41.7% and 4.5% and in the female group in 51.0% and 7.5% of malignant and benign PEs, respectively. A statistically significant correlation between hMAM and malignancy was found in the entire population (DOR=14.68, P<0.001) and in the male (DOR=15.00, P<0.001) or female (DOR=12.77, P<0.001) groups. hMAM RT-PCR increased the diagnostic rate of malignant PEs as it allowed us to detect as malignant 32.1% of cytologically negative PEs. In female patients the positivity of hMAM indicated with higher probability (50.8%) the origin of PEs from breast cancer but lower probability from LC (17%), MM (9.4%), or other cancers (15.1%), whereas in male patients it indicated with similar probability (about 40%) the origin from LC or MM. Our results suggest that hMAM RT-PCR may provide information both in the diagnosis of PE and in the search for the primary site of neoplasia, either in male or female patients.