In biological fluids, hCG and its free alpha- (hCG alpha) and beta-subunits (hCG beta), occur in multiple forms. These various forms differ at the molecular level primarily in glycosylation, but also differ in protein backbone modifications corresponding to the urinary low molecular weight fragment of the hCG beta-subunit (beta-core fragment). This microheterogeneous nature can be demonstrated by isoelectric focusing in which variants are separated into bands with different isoelectric points (pI). To determine whether such isoelectric variants differ in antigenicity and consequently might escape immunoassay detection due to overspecificity of monoclonal antibodies (MCA), urinary pregnancy hCG (NIH, CR123) and tumor hCG preparations, such as a tumor-specific acidic variant of hCG (hCGav) and the hCG beta-core fragment, were separated by isoelectric focusing in the absence or presence of 8 M urea, or by sodium docedyl sulfate-polyacrylamide gel electrophoresis and enzymatically immunostained using an MCA panel directed against 17 different hCG epitopes. MCA against 14 different epitopes accessible on holo-hCG recognized all pI variants of pregnancy holo-hCG or tumor-derived hCGav, as was true for the three MCA recognizing epitopes hidden on holo-hCG but accessible on the free subunits after hCG dissociation by urea. We conclude that each individual pI-isoform of holo-hCG and its free subunits expresses the entire set of epitopes recognized by our MCA panel. The carbohydrate moieties that form a biochemical basis for hCG heterogeneity seem to be neither of major antigenic relevance, nor are they structurally related to any particular epitope. Thus, various glycosylation forms of hCG, hCG alpha, hCG beta, and hCG beta-core in normal as well as in pathological samples should safely be detectable and measureable by immunoassays employing MCA with appropriate subunit specificity.