The hCG beta-subunit contains a carboxy-terminal extension bearing four serine-linked oligosaccharides [carboxy-terminal peptide (CTP)], which is important for maintaining its longer half-life compared with the other glycoprotein hormones. Previously, we enhanced the in vivo half-life of FSH by fusing the CTP to the carboxy end of FSH beta coding sequence. The alpha-subunit is common to the glycoprotein family. We constructed alpha-subunit CTP chimeras, since such analogs with the appropriate O-linked glycosylation and conformation would increase the in vivo stability of the entire glycoprotein hormone family. Two chimeras were constructed using overlapping polymerase chain reaction mutagenesis: a variant with CTP at the carboxy end and another analog with the CTP at the N-terminal region of the subunit, between amino acids 3 and 4. The latter design was based on models showing that the amino-terminal region of alpha is not involved in assembly with the beta-subunit, nor is it essential for receptor binding and signal transduction. These chimeras were cotransfected with the hCG beta gene into Chinese hamster ovary cells. The chimeras were secreted and combined efficiently with the CG beta-subunit, comparable to the wild type alpha-subunit. CG dimers containing the alpha-subunit chimera with CTP at the carboxy end of the subunit had a much lower binding affinity for the hLH-hCG receptor in vitro, whereas the binding of the dimer containing the CTP at the amino-terminal end of the subunit was similar to wild type hCG. Furthermore, the in vivo activity of this analog was enhanced significantly. Moreover, regardless of the two insertion points in the alpha-subunit, the CTP sequence was O-glycosylated. These data suggest that the entire signal for O-glycosylation is primarily contained within the CTP sequence and is not dependent on the flanking regions of the recipient protein. The transfer of CTP to the alpha-subunit of hCG results in an agonist with prolonged biological action in vivo. These data further support the rationale for using the CTP as a general target to increase the potency of bioactive glycoproteins.