A novel antifungal peptide (termed as Anafp) was isolated from the culture supernatant of the filamentous fungi, Aspergillus niger. The whole amino acid sequence of Anafp was determined and the peptide was found to be composed of a single polypeptide chain with 58 amino acids including six cysteine residues. The peptide shows some degree of sequence homology to a cysteine-rich antifungal peptides reported from the seeds of Sinapis alba and Arabidopsis thaliana or the extracellular media of Aspergillus giganteus and Penicillium chrysogenumsome. Cysteine-spacing pattern of Anafp was similar to that of the antifungal peptide from Penicillium chrysogenum. The Anafp exhibited potent growth inhibitory activities against yeast strains as well as filamentous fungi at a range from 4 to 15 microM. In contrast, Anafp did not show antibacterial activity against Escherichia coli and Bacillus subtilis even at 50 microM.