A novel ribosome-inactivating protein with a molecular weight of 20 kDa was isolated from fruiting bodies of the mushroom Hypsizigus marmoreus. The isolation procedure entailed ion exchange chromatography on CM-cellulose, affinity chromatography on Affi-gel Blue Gel and ion exchange chromatography on Mono Q. The protein designated hypsin demonstrated an inhibitory action against mycelial growth in various fungal species including Mycosphaerella arachidicola, Physalospora piricola, Fusarium oxysporum, and Botrytis cinerea with an IC50 of 2.7, 2.5, 14.2, and 0.06 muM, respectively. Translation in the rabbit reticulocyte lysate system was inhibited with an IC50 of 7 nM and HIV-1 reverse transcriptase activity was inhibited with an IC50 of 8 muM. Antiproliferative activity against mouse leukemia cells and human leukemia and hepatoma cells was observed. About 60% of the translation-inhibitory activity was retained after heating at 100 degreesC for 10 min. No loss of translation-inhibitory activity occurred after brief treatment with trypsin.