AbstractInsulin‐like peptide 3 (INSL3) is a peptide hormone belonging to the relaxin‐insulin superfamily of peptides that plays important roles in testes descent, oocyte maturation and the control of male germ cell apoptosis. These actions are mediated via a specific G‐protein coupled receptor, LGR8. Previous structure–activity studies have shown that the key binding site of INSL3 is situated within its B‐chain. Recent studies in our laboratory have led to the identification of a cyclic peptide mimetic 2 of the INSL3 B‐chain, which we have shown to compete with the binding of [33P]‐relaxin to LGR8 expressed in HEK293T cells, and to inhibit cAMP‐mediated signaling in these cells, i.e. it is an antagonist of INSL3. In order to further define the structure–activity relationships of cyclic analogues of the INSL3 B‐chain, we used a structure‐based approach to design a series of cyclic, disulfide‐constrained INSL3 B‐chain mimetics. To do this, we first created a model of the 3D structure of INSL3 using the crystal structure of human relaxin as a template. This model of INSL3 was then used as a template to design a series of disulfide‐constrained mimetics of the INSL3 B‐chain. The peptides were synthesized by solid‐phase peptide synthesis using pseudoproline dipeptides to improve the synthesis outcome. Of the seven prepared INSL3 B‐chain mimetics, three compounds were found to have partial displacement activity, while four were able to completely displace [33P]‐relaxin from LGR8, including compounds that were markedly shorter than compound 2. The best of these, mimetic 6, showed significantly greater affinity for LGR8 than compound 2, but still displayed around 1000‐fold less affinity for LGR8 than native INSL3. Analysis of selected mimetics for their α‐helical content using circular dichroism (CD) spectroscopy revealed that, generally, the mimetics showed less than expected helicity. The inability of the compounds to display true native INSL3 structure is likely contributing to their reduced receptor binding affinity. We are currently examining alternative INSL3 B‐chain mimetics that might better present key receptor binding residues in the native INSL3‐like conformation. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.