The flowers of the solanaceous plants ornamental tobacco (Nicotiana alata) and petunia (Petunia hybrida) produce high levels of defensins during the early stages of development. In contrast to the well-described seed defensins, these floral defensins are produced as precursors with C-terminal prodomains of 27 to 33 amino acids in addition to a typical secretion signal peptide and central defensin domain of 47 or 49 amino acids. Defensins isolated from N. alata and petunia flowers lack the C-terminal domain, suggesting that it is removed during or after transit through the secretory pathway. Immunogold electron microscopy has been used to demonstrate that the N. alata defensin is deposited in the vacuole. In addition to the eight canonical cysteine residues that define the plant defensin family, the two petunia defensins have an extra pair of cysteines that form a fifth disulfide bond and hence define a new subclass of this family of proteins. Expression of the N. alata defensin NaD1 is predominantly flower specific and is most active during the early stages of flower development. NaD1 transcripts accumulate in the outermost cell layers of petals, sepals, anthers, and styles, consistent with a role in protection of the reproductive organs against potential pathogens. The floral defensins inhibit the growth of Botrytis cinerea and Fusarium oxysporum in vitro, providing further support for a role in protection of floral tissues against pathogen invasion.