From unripe fruits of Bromelia hieronymi Mez (Bromeliaceae), a partially purified protease preparation was obtained by acetone fractionation of the crude extract. Purification was achieved by anionic exchange chromatography (FPLC) on Q-Sepharose HP followed by cationic exchange chromatography (SP-Sepharose HP). Homogeneity of the new enzyme, named hieronymain II, was confirmed by SDS-PAGE and mass spectroscopy (MALDI-TOF-TOF). The molecular mass of was 23,411 Da, and maximum proteolytic activity (more than 90% of maximum activity) was achieved at pH 7.5-9.0 on casein and at pH 7.30-8.3 on Z-Phe-Arg-p-nitroanilide. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine. The N-terminal sequence of hieronymain II (AVPQSIDWRVYGAV) was compared with those of 12 plant cysteine proteases which showed more than 70% of identity. Kinetic enzymatic assays were made on Z-Phe-Arg-p-nitroanilide (Km = 0.72mM, kcat = 1.82 seg(-1) , kcat/ Km = 2.54seg(-1) mM(-l)). No detectable activity could be found on PFLNA or Z-Arg-Arg-p-nitroanilide.