Human chorionic gonadotropin (hCG) is a member of the family of glycoprotein hormones containing a common α-subunit and distinct β-subunits that confer hormonal specificity. hCG binds to the relatively large ectodomain of the human luteinizing hormone receptor (hLHR), a member of the G protein-coupled receptor superfamily, leading to increased intracellular production of cAMP. Using protein engineering, two miniaturized versions of hCGβ have been separately fused to the N-terminus of the α-subunit to give N-des[1-91]hCGβ-α-C and N-des[1-91,110-114]hCGβ-α-C, i.e. fusion proteins of the hCGβ determinant loop (extended to include the complete seat belt and carboxy-terminal peptide) coupled to the α-subunit. Bioactivity of these single-chain gonadotropin analogs was assessed in two systems following transient transfections into HEK 293 cells and subsequent cAMP measurements. In one, each mini-β-α cDNA was fused to that of hLHR and transfected into cells to create yoked miniaturized hCG-hLHR complexes; in the other, the cDNA of each single chain mini-β-α was co-transfected with that of hLHR in an effort to produce non-covalent miniaturized hCG-hLHR complexes. Using yoked hCG-hLHR and hLHR as positive and negative controls respectively, expression of each mini-hCG-hLHR complex was confirmed using antibody and ligand binding assays. The two mini-hCGs led to minimal activation of hLHR, suggesting weak intrinsic activity of the mini-β-α fusion proteins. These results suggest that potent agonists and antagonists will require the presence of other portions of hCGβ in addition to the determinant loop/seat belt.