To investigate the possibility that the human pituitary gland secretes CG, we used a highly specific, two-site, double monoclonal immunoradiometric assay to measure CG in the medium in which the dispersed cells of pituitary glands from human fetuses of 20-24 weeks gestation were cultured. The cross-reactivity of immunopurified human LH in the CG assay was less than 0.03%. LH was also measured by a double monoclonal immunoradiometric assay. Secretion of CG by the cultured fetal pituitary cells was readily detectable, although in gradually decreasing amounts, for the 11 days of culture. LH secretion paralleled CG secretion and was much greater in magnitude. Pituitary cells from female fetuses generally secreted more CG as well as more LH than those from male fetuses. Dilutions of the medium showed that the secreted CG exhibited parallelism with the CG standard. Chromatofocusing of the medium across a pH 6-9 gradient yielded several peaks of LH immunoreactivity between pH 6.5 and 8.5, but no peaks of CG. Chromatofocusing of the medium across a pH 3-7 gradient yielded peaks of CG, but not LH, between pH 4.0-5.5. These data indicate that CG immunoreactivity, distinct and separable from LH immunoreactivity, is secreted by the dispersed cells of fetal human pituitary glands.