TSH and the gonadotropins, FSH, LH, and CG are a family of heterodimeric glycoprotein hormones composed of a common α-subunit noncovalently linked to a hormone specific β-subunit. Assembly of α- and β-subunits is essential for hormone-specific posttranslational modifications, receptor binding, and bioactivity. Structure-function studies of TSH and gonadotropins using site- directed mutagenesis can often affect folding, assembly, and secretion of the hormone. To circumvent these difficulties, recently, the gonadotropin heterodimers were converted to single chains. Here we converted the hTSH heterodimer to a biologically active single chain by genetically fusing the amino terminal end of the common α-subunit to the carboxyl terminal end of hTSHβ in the presence or absence of hCGβ carboxyl terminal peptide (CTP), which was used as a linker. Wild-type hTSH and the single chains were expressed in Chinese hamster ovary (CHO) cells, and they were efficiently secreted. Although the secretion rate of the single chain was 3-fold higher than that of hTSH wild-type. Moreover, the secretion of the single chain in the presence of the CTP linker was dramatically increased. On the other hand, receptor binding and in vitro bioactivity of the single chains were similar to that of hTSH wild-type. These data indicate the potential of the single chain approach to further investigate structure-function relationships of TSH.