The primary structure of ovine Leydig cell insulin-like peptide (Ley I- L) was recently deduced from the corresponding cDNA sequence. It consists of two peptide chains and three disulphide bonds in an arrangement similar to both relaxin and insulin. As in relaxin B-chain, an Arg-X-X-X-Arg sequence exists within the Ley I-L B-chain although it is located four residues towards the C-terminus from the corresponding position within relaxin. This sequence of amino acids is known to be essential for relaxin biological activity and its presence in Ley I-L suggested that the peptide might possess a relaxin-like function. Ovine Ley I-L was assembled by Fmoc-solid-phase synthesis of the separate chains followed by their combination in solution at high pH. The purity and identity of the chain-combined peptide was confirmed by chemical characterization including mass spectrometry. At physiological concentrations, the peptide was shown not to possess relaxin-like activity in the rat isolated atrial chronotropic and inotropic assay. This strongly suggests that Ley I-L is not a relaxin in the sheep. In order to explore further a possible structural relationship between Ley I-L and relaxin, we prepared a synthetic analogue of ovine Ley I-L containing a single replacement of B-chain residue 12, His, with Arg. This was found to possess significant relaxin-like chronotropic and inotropic activity demonstrating that the tertiary structure of Ley I-L is similar to that of relaxin and highlighting the key requirement for the five-residue sequence, Arg-X-X-X- Arg, to be present in position B12-16 for characteristic relaxin activity.