Abstract Background: The currently used standards for human chorionic gonadotropin (hCG) and its α and β subunits (hCGα and hCGβ) contain substantial amounts of contaminating variants of hCG and other impurities. Furthermore, some partially degraded forms of hCG and its subunits have become of potential clinical importance, e.g., “nicked” forms of hCG (hCGn) and hCGβ (hCGβn), which contain cuts in the peptide backbone between amino acids 44–45 or 47–48 in hCGβ, and a fragment of hCGβ (hCGβcf) consisting of amino acids 6–40 and 55–92 bound together by disulfide bridges. The IFCC appointed a working group with the aim of preparing new standards for hCG and related substances to improve standardization of their immunoassays. Methods: Large amounts of hCG and its subunits as well as of hCGn, hCGβn, and hCGβcf were prepared by previously developed purification methods in combination with hydrophobic interaction chromatography and reversed-phase HPLC. Each preparation was characterized on the basis of amino acid and sequence analyses, carbohydrate composition, and electrophoretic patterns. Immunoassays for relevant contaminating proteins were also performed. Results: The major preparations were homogeneous and free of contaminating proteins. Concentrations of the final preparations were determined by amino acid analysis. Conclusions: Calibrated in substance concentrations (mol/L) based on amino acid analyses, these preparations will facilitate improved standardization of immunoassays for hCG and its metabolites. The six preparations have now been established by the WHO as new 1st Reference Reagents for immunoassays with the following codes: hCG 99/688, hCGβ 99/650, hCGα 99/720, hCGn 99/642, hCGβn 99/692, and hCGβcf 99/708. In contrast to the 3rd International Standard (75/537), the clinically most important Reference Reagent for hCG (99/688) contains no hCGn and negligible amounts of free subunits.