Cytomorphologic and immunocytologic examination alone provide only limited sensitivity for the detection of metastatic carcinoma cells in many cases of serous effusions. The specificity of conventional reverse transcription-polymerase chain reaction (RT-PCR) methods for detection of epithelial gene transcripts is low owing to the ectopic expression of many such genes in nonepithelial cells. For the detection of metastatic carcinoma cells, we describe a quantitative real-time RT-PCR assay for GA733-2 messenger RNA encoding an epithelial glycoprotein (EGP-2) that binds to the monoclonal antibody BerEP4. With serial dilutions of BerEP4-positive SK-BR-3 breast carcinoma cells, the RT-PCR assay was able to detect 10 carcinoma cells in a background of 10(6) lymphoid cells compared with 10(2) to 10(3) carcinoma cells detectable by immunocytologic examination. We analyzed 51 serous effusions, including 25 malignant metastatic effusions, by the real-time RT-PCR assay. Receiver operating characteristic curve analyses demonstrated a sensitivity of 96% corresponding to a specificity of 98% for a correct diagnosis of metastatic effusions. These results provide evidence that the GA733-2 real-time RT-PCR assay is a specific and sensitive tool for the detection of metastasis of BerEP4-positive primary tumors in serous effusion specimens.