The β subunit of human chorionic gonadotropin (hCG) contains at its carboxy terminus an extension of 29 amino acids not found in the β subunits of the other glycoprotein hormones. This region provides the sites of attachment of four serine-linked oligosaccharide chains. We have examined the synthesis of this subunit in a cell-free translation system derived from Krebs II ascites tumor cells. The primary translation product was found to undergo a temperature-de-pendent posttranslational modification which resulted in an increase in apparent molecular weight of 2000 on sodium dodecyl sulfate gel electrophoresis. This modification was specific for the β subunit of hCG, since no changes were observed for the β subunit of bovine luteinizing hormone or for the a subunits of either hormone. The increase in molecular weight occurred in the absence of microsomal membranes and was not due to the addition of N-linked carbohydrate. An identical shift was observed when pre-hCG β was incubated with extracts of human placenta. The site of modification was localized by fingerprint analysis to a carboxy-terminal tryptic peptide which contains two of the four O-glycosylated serine residues in the mature form of the subunit. The modified protein was resistant to oligo-saccharidase digestion and β-elimination, indicating that it does not contain O-linked oligosaccharides of the type found on mature hCG β. These results demonstrate that a specific modification of the carboxy-terminal segment of hCG β synthesized in vitro occurs in the absence of O-linked glycosylation.