Puroindolines are two small, basic cysteine-rich proteins isolated from Triticum aestivum seeds and characterized by a tryptophan-rich domain. They form the molecular basis of wheat grain hardness and display antimicrobial activity that may contribute to plant defence. Their antimicrobial activity is presumed to be due to their hydrophobic tryptophan-rich domain. However, little is known about their mode of action and there is no in vivo evidence that the binding of puroindolines to membranes is mediated by their tryptophan-rich domain. In this study, using a yeast complementation assay, we showed that puroindolines interact with the Saccharomyces cerevisiae plasma membrane. By site-directed mutagenesis of their tryptophan-rich domain, we determined that two tryptophan residues (W41 and W44) are mandatory for interaction of puroindoline-a with the yeast membrane whereas interaction of puroindoline-b depends on lysine residues. These results highlight that other residues than tryptophan play a critical role in the interaction of puroindolines with membranes, and probably their affinity for lipids and antimicrobial activities