Concentrations of human choriogonadotropin (hCG) and its free beta subunit (beta hCG) were measured in serum by highly sensitive and specific time-resolved immunofluorometric assays (IFMAS). The results were confirmed by completely separating beta hCG and hCG by a novel method based on hydrophobic-interaction chromatography. We used three monoclonal antibodies in two different combinations. In both assays an antibody reacting with both free beta hCG and with intact hCG was immobilized onto the wall of a microtiter strip well. For assay of intact hCG we used as the indicator antibody an antibody against the alpha subunit, labeled with a europium chelate. For assay of beta hCG we used an indicator antibody that reacted only with the free beta subunit. hCG cross-reacted in the assay of beta hCG by 0.6%. Quantifying hCG in serum after in vitro fertilization showed that, seven to eight days after embryo transfer, the hCG concentration started to increase, thereafter increasing with a doubling time of 1.9 days during the following three weeks. hCG concentrations in serum peaked six to 10 weeks later, corresponding to eight to 12 weeks after the last menstrual period. Throughout pregnancy, measurable amounts of beta hCG were present in serum. The highest beta hCG/hCG ratio (maximum 7.3%, median 3.0%) was observed during early gestation. During the fourth to 13th weeks after the last menstrual period the ratio of beta hCG/hCG decreased gradually, being 1.0% during the second and third trimesters.