The sequence of the puroindoline-b gene from 15 Australian wheat cultivars was determined. Sequence variation was observed in the WPTKWWKGGCE motif of the deduced puroindoline-b protein sequence. Previously, it has been suggested that this sequence is crucial in determining grain hardness. In this study, no correlation was found between the variation in this sequence and the hardness or softness of the cultivar. The amounts of puroindoline-a and puroindoline-b protein in a selection of hard and soft Australian wheat cultivars were also determined using ELISA techniques. Both soft and hard cultivars had variable amounts of puroindoline-a and puroindoline-b. In particular, it is notable that the hard cultivars Cook and Diaz contained high amounts of puroindoline-a and puroindoline-b and also contained the puroindoline-b sequence previously reported to be associated with grain softness. These results suggest that if the puroindoline proteins are involved in determining grain softness or hardness they do so as part of a multi-component mechanism.