Human chorionic gonadotrophin (hCG), like other members of the glycoprotein hormone family, contains a common α subunit and a hormone-specific β subunit. The latter is a 145 amino acid residue polypeptide with six sites of glycosylation. Positions 2 and 104 are occupied by basic amino acid residues in the 12 known amino acid sequences of mammalian β subunits from CG and LH, a related gonadotrophin that acts through the same receptor. Lysine residues are found in both these positions in hCG-β. Using site-directed mutagenesis, each of these two lysines in hCG-β was replaced with glutamic acid. The mutant and wild-type cDNAs were subcloned into a eukaryotic expression vector, which was then transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for the bovine α subunit. Holoprotein formation occurred with each of the two heterologous gonadotrophin mutants, i.e. the bovine α subunit bound to hCG-β (Glu2) and to hCG-β (Glu104), as well as with the control, i.e. the bovine α subunit bound to the hCG-β wild-type subunit. In two in-vitro assays, one a competitive binding assay with 125I-labelled hCG as bound ligand and the other based on stimulation of progesterone production in a transformed murine Leydig cell line, MA-10, both the heterodimers containing a mutant β subunit exhibited bioactivity, but their potencies were lower than that of the bovine α subunit bound to the hCG-β wild-type subunit. These results suggest that the basic amino acid residues at positions 2 and 104 in hCG-β participate, either directly or indirectly, in receptor binding.