Relaxin is a peptide with various reproductive and nonreproductive functions. The site for the peptide-receptor interaction contains two arginines (Arg) and an isoleucine (Ile) or valine (Val) residue in the B-chain with a configuration of -Arg-X-X-X-Arg-X-X-Ile/Val-X-. The sheep insulin-like peptide 3 (INSL3), a structural homologue of relaxin, also contains the n, n+4 arginines in the B-chain but they are displaced towards the carboxyl terminus by four residues (-X-X-X-X-Arg-X-X-Val-Arg-). Human INSL3 increases the activity of human relaxin in mouse bioassays. Here, we investigated whether sheep synthetic INSL3 affects the relaxin activity in rat atria. INSL3 lacked relaxin-like agonist activity but blocked the activity of relaxin and competed for relaxin binding sites at high concentrations. We also synthesized analogues of INSL3, with amino acid substitutions in the arginine-binding region. Analogues A, D and E, which have the arginines in positions identical to relaxin, showed weak relaxin-like agonist activity. These results suggest that other sites in the relaxin molecule are involved in high-affinity peptide-receptor interaction for the production of the relaxin biological responses.