The wheat starch 15-kDa protein (called grain softness protein or GSP) consists of a major polypeptide and several minor polypeptides. An antiserum raised against GSP was used to screen a wheat cDNA library. A cDNA family encoding approximately 15-kDa proteins that included a heptapeptide sequence previously isolated from protease digests of GSP was identified. A partial cDNA was used in a prokaryotic expression system to produce a fusion protein which reacted strongly against the original anti-GSP serum. A new antiserum raised against the fusion protein produced a weak reaction against a 15-kDa polypeptide extracted from wheat seeds. The results suggest that the proteins encoded by the cDNA family form a minor component of the mixture of 15-kDa polypeptides defined as GSP. RNA complementary to the cDNAs could be extracted from both soft and hard wheat grains from about half-way through grain filling. The encoded proteins are novel members of the 2S superfamily of seed proteins, a diverse family of proteins which maintain a characteristic framework of cysteine residues. The deduced proteins show the highest similarity to the oat 16-kDa avenin and to wheat puroindoline (a lipid-binding 15-kDa protein from wheat). Review of previously published data shows that puroindoline is also closely related to the major polypeptide of GSP, suggesting that the lipid-binding properties of GSP polypeptides may influence grain softness.