The isolation of a protein designated mollisin, with an N-terminal sequence manifesting some similarity to thaumatin-like proteins (TLPs), and possessing a molecular mass of 28 kDa which is higher than those of TLPs, is reported herein from the seeds of the chestnut Castanea mollisima. The protein was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and Mono S. Mollisin exhibited a molecular mass of 28 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as well as in gel filtration on Superdex 75 by fast protein liquid chromatography. The protein inhibited mycelial growth in Fusarium oxysporum, Mycosphaerella arachidicola and Physalospora piricola, with an IC (50) of 0.83 microM, 6.48 microM and 9.21 microM, respectively. Mollisin displayed a higher antifungal potency than French bean and kiwi fruit TLPs toward F. oxysporum and M. arachidicola. The antifungal activity of mollisin was unaffected by incubation at 40 degrees C for 10 minutes, underwent a decline after incubation at 60 degrees C, and was completely abolished after treatment at 80 degrees C. Mollisin exhibited a more potent inhibitory activity on HIV-1 reverse transcriptase than kiwi fruit TLP.