The concentration of immunoreactive alpha and beta subunits of human LH (hLH) and human FSH (hFSH) were measured in unfractionated serum throughout normal menstrual cycles, and when GnRH was administered at about midcycle. The RIA subunit systems employed were sufficiently sensitive and specific to measure basal levels of free alpha-hLH, beta-hLH, and alpha-hFSH. The beta-hFSH system lacked sufficient sensitivity to measure beta-hFSH in unfractionated serum. In normal cycles, alpha-hLH, beta-hLH and alpha-hFSH were shown to circulate in the free form with highest concentrations occurring at the time of midcycle peak of LH and FSH. The concentration of subunits during the follicular phase were not significantly different from those observed during the luteal phase of the cycle. In response to GnRH administration during the periovulatory phase of the cycle, the increases in circulating free alpha-hLH, beta-hLH, and of alpha-hFSH subunits were directly related to the increases in LH and FSH. Other investigators have shown that increases in circulating levels of free subunits are due to secretion from the pituitary gland rather than from peripheral dissociation of intact hormone. Therefore, it can be assumed that the increase in immunoreactive alpha-hLH, beta-hLH, and of alpha-hFSH subunits observed in this study at midcycle represent release of free subunits in response to endogenous or exogenous GnRH administration.