The expression of human chorionic gonadotropin (hCG), its subunits, and fragments on the cell membrane of cultured human cancer cells was investigated using a flow cytometric method. This method uses living cells; a double-antibody reaction; a flow cytometer with an argon laser, standard settings, and filters for fluorescein isothiocyanate; commercially available software; the American Type Culture Collection (ATCC) CCL 2 HeLa cell line as cell control and overall quality control; polyclonal rabbit antisera raised against the hCG dimer, its alpha subunit (hCG alpha), and its beta subunit (hCG beta); and a panel of monoclonal antibodies (MoAb) recognizing different epitopes on the intact hCG molecule, its subunits, and fragments. The purified immunoglobulin G fractions from the polyclonal antisera were used to estimate the total expression of the membrane-associated glycoproteins; the MoAb were used to detect the expression of epitopes of the hCG dimer, its subunits, and fragments. The results of the analyses done on cells from 74 established cancer cell lines of different types and origins (including 52 carcinomas, 10 sarcomas, 4 leukemias, 6 lymphomas, and 2 retinoblastomas) showed variable degrees of reactivity in a great percentage of cells in all cell lines studied with MoAb directed against different conformational epitopes of intact hCG (hCG-holo), hCG beta, hCG beta-free, the carboxy terminal peptide (CTP) of hCG beta, and an epitope of hCG alpha. The expression of the membrane-associated epitopes of hCG and its subunits was found to be a phenotypic marker characteristic of all evaluated cultured human cancer cell lines, irrespective of their type or origin. There were, however, quantitative and qualitative differences in the expression of the different epitopes. Thus, hCG beta, free and as part of hCG-holo, recognized by the MoAb against hCG beta-CTP, was expressed by a high percentage of cells of most cell lines. There was great variability in the expression of hCG-holo, recognized by MoAb B109. For this reason some groups of cancers expressed larger amounts of incompetent hCG alpha and/or hCG beta than others. Cell lines derived from adenocarcinomas of the lung were the only exception to this general finding; the expression of small amounts of hCG-holo was caused by a low degree of hCG alpha synthesis.