Levels of hCG alpha beta dimer, free alpha-subunit and free beta-subunit were measured in pregnancy sera. Dimer and free alpha were quantitated by radioimmunoassays (RIAs) using specific polyclonal antisera. Free beta was quantitated both by monoclonal anti-beta RIA and by polyclonal anti-beta RIA following the complete adsorption of cross-reacting hCG by immobilized alpha-antisera. Consistent with the findings of other laboratories, hCG levels in pregnancy sera peaked at around 10 weeks after the last menstrual period (post LMP), and declined thereafter. Free alpha levels rose as hCG levels declined and accounted for 30-40% of total serum alpha in the third trimester. Although free beta accounted for only a small proportion of the total beta-subunit at the time of the hCG peak and thereafter (2.4-3.6%), in early pregnancy serum samples, 4-6 weeks post LMP, when hCG was generally first detected, an average of 16% free beta was detected. At this time, the higher the hCG level (20-2000 ng/ml), the lower the percent free beta (54-3%). Thus, the free beta portion started high and declined prior to the hCG peak; the free alpha portion increased thereafter. To explain these findings, we propose a two phase regulation of hCG dimer formation. Up to the time of the hCG peak, supplies of alpha-subunit are limiting (hence the presence of free beta). Thereafter, beta-subunit levels drop, restricting dimer formation and leaving uncombined alpha.