An 18.9 kDa antifungal protein designated juncin was isolated from seeds of the Japanese takana (Brassica juncea var. integrifolia). The purification protocol employed comprised anion-exchange chromatography on Q-Sepharose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration on Superdex 75. Juncin was adsorbed on Affi-gel blue gel and SP-Sepharose but unadsorbed on Q-Sepharose. The protein exhibited antifungal activity toward the phytopathogens Fusarium oxysporum, Helminthosporium maydis, and Mycosphaerella arachidicola with IC(50) values of 13.5, 27, and 10 μM, respectively. It was devoid of mitogenic activity toward splenocytes and nitric oxide inducing activity toward macrophages. It inhibited the proliferation of hepatoma (HepG2) and breast cancer (MCF7) cells with IC(50) values of 5.6 and 6.4 μM, respecitvely, and the activity of HIV-1 reverse transcriptase with an IC(50) of 4.5 μM. Its N-terminal sequence differed from those of antifungal proteins that have been reported to date. Compared with Brassica campestris and Brassica alboglabra antifungal peptides, juncin exhibits a different molecular mass and N-terminal amino acid sequence but similar biological activities.