Relaxin is an insulin-like peptide consisting of two separate chains (A and B) joined by two inter- and one intrachain disulfide bonds. Binding to its receptor requires an Arg-X-X-X-Arg-X-X-Ile motif in the B-chain. A related member of the insulin superfamily, INSL3, has a tertiary structure that is predicted to be similar to relaxin. It also possesses an Arg-X-X-X-Arg motif within its B-chain, although this is displaced by four amino acids towards the C-terminus from the corresponding position within relaxin. We have previously shown that synthetic INSL3 itself does not display relaxin-like activity although analogue (Analogue A) with an introduced arginine residue in the B-chain giving it an Arg cassette in the exact relaxin position does possess weak activity. In order to identify further the structural features that impart relaxin function, solid phase peptide synthesis was used to prepare three additional analogues for bioassay. Each of these contained point substitutions within the arginine cassette. Analogue D contained the full human relaxin binding cassette, Analogue G consisted of the native INSL3 sequence containing an Arg to Ala substitution, and Analogue E was a further modification of Analogue A, with the same substitution. Each analogue was fully chemically characterized by a number of criteria. Detailed circular dichroism spectroscopy analyses showed that the changes caused little alteration of secondary structure and, hence, overall conformation. However, each analogue displayed only weak relaxin-like activity. These results indicate that while the arginine cassette is vital for relaxin-like activity, there are additional, as yet unidentified structural requirements for relaxin binding.