Human CG (hCG) is the hormone associated with the maintenance of pregnancy. Although three related glycoprotein hormones, LH, hFSH, and hTSH, are secreted by the pituitary, HCG is the only one of this family of glycoprotein hormones that is produced by the placenta in primates to maintain the steroid hormone secretions of the corpus luteum. Although hCG is not considered to be a pituitary hormone, hCG-like immunoreactive materials have been reported in pituitary tissue, blood, and urine from healthy nonpregnant individuals for 2 decades, but it was never isolated. We now report the purification and characterization of pituitary hCG from acetone-preserved human pituitary glands. After gell permeation chromatography, the fractions containing hCG molecules were pooled and purified by immunoaffinity chromatography using antibodies to the COOH-terminal region of hCGbeta. Amino acid analyses, amino-terminal sequence analyses, as well as mass spectrometric studies gave similar results for both pituitary hCG and hCG purified from the urine of pregnant women. Analyses for sulfate and sialic acid contents demonstrated that pituitary hCG contained both sulfate and sialic acid. In vitro biological activity of pituitary hCG indicated that it was 50% as active as hCG purified from the urine of pregnant women in cAMP assays.