A novel antifungal protein, M-r = ca. 40 kDa, was isolated from pumpkin rind and designated Pr-1. When purified by anion exchange chromatography and HPLC, it inhibited growth of several fungi including Botrytis cinerea, Fusarium oxysporum, Fusarium solani and Rhizoctonia solani, as well as the yeast, Candida albicans, at 10-20 mu M. It did not inhibit growth of Escherichia coli or Staphylococcus aureus even at 200 mu M. Laser scanning microscopy of fungal cells exposed to rhodamine-labeled Pr-1 revealed that the protein accumulated and was localized on the cell surface. Uptake of the vital stain, SYTOX Green, was enhanced when fungal conidia were treated with Pr-1 suggesting that the protein has membrane permeabilization activity. Pr-1 was thermostable at 70A degrees C and did not lyse human red blood cells at 128 mu M suggesting that the protein may be useful as an antifungal agent with little, if any human cytotoxicity.