We have isolated a cDNA clone for the antifungal protein, CaAFP, from hot pepper, Capsicum annuum L. Its open reading frame encodes 85 amino acids, including 8 cysteine residues. CaAFP consists of three domains: a signal peptide, a chitin-binding domain, and a C-terminal peptide domain. The deduced amino acid sequence of the chitin-binding domain shows 92% and 85% similarity to the same domain from PnAMPs and hevein, respectively. Southern blot analysis indicated that CaAFP is present as a single copy, while the northern blots revealed that the clone is highly expressed in the leaves and flower buds, but not in the roots. However, wounding treatments and chemicals generally known to induce PR proteins did not stimulate its expression. In situ hybridization also showed that CaAFP is expressed in the parenchyma cells of the floral sepals. As seen in our functional analysis, this clone was expressed in Escherichia coli, and the fusion protein was purified using nickel-affinity column chromatography. This purified AFP fusion protein inhibited spore germination and appressoria formation in several plant pathogenic fungi, including Fusarium oxisporum and Colletotrichum gloeosporioides. Our results suggest CaAFP is an antifungal protein that defends developing seeds against pathogen invasion while also having a specific biological role during floral development.