Peptide hormones that modulate insulin secretion have been recognized to have therapeutic potential, with peptides such as amylin (pramlintide acetate, Symlin) and exendin-4 (exenatide, Byetta) now commercially available. Preptin is a peptide that has been shown to increase insulin secretion in vitro and in vivo. Here, we describe the first chemical synthesis and analysis of a short series of preptin analogues based on the rat preptin sequence. Phe 21 in the preptin sequence was substituted with the non-protein amino acids D-Phe, D-Hphe, 3-aminobenzoic acid and 1-aminocyclooctane-1-carboxylic acid, which rendered the preptin analogues resistant to chymotryptic protease hydrolysis at this position. Substitution of Phe 21 with these non-protein amino acids did not abrogate the insulin secretory effect of preptin, with analogues showing a similar dose-dependent effect on insulin secretion from βTC6-F7 mouse β-cells in both the presence and absence of glucose as unmodified rat preptin. Further studies on the stability of the preptin analogues and their effect on insulin secretion are in progress.