In contrast to the cellular receptors for insulin and insulin-like growth factors that are known to be protein tyrosine kinases, those of both insulin 3 and relaxin have recently been identified as being members of the leucine-rich repeat-containing G-protein coupled receptor (LGR) family, LGR8 and LGR7, respectively. This has prompted an examination into the possibility that they might also be specific for another member of the insulin superfamily, namely, insulin 4. Towards this aim, a two-chain peptide corresponding to the predicted primary structure of insulin 4 was prepared by solid phase synthesis. As conventional aeration and combination of the two S-reduced chains in solution at high pH failed to produce target product, selectively S-protected A- and B-chains were prepared followed by stepwise, individual formation of each of the three disulfides, one intramolecular within the A-chain and two intermolecular. Chemical characterization confirmed the purity and identity of the synthetic insulin 4 analogue. However, secondary structural analysis indicated that the peptide was devoid of tertiary conformation suggesting that the native peptide may well be either significantly longer in length or is similar to insulin-like growth factor I or II in that it is a single chain product. Screening of the synthetic analogue for activation of transfected cells bearing LGR7, and LGR7 splice variant or LGR8 failed to identify a specific interaction. Thus, the in vivo structural identity of insulin 4 and its receptor (if any) as well as its potential function remains unknown.