Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain c-Ⅱ) has been iso-lated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain c-Ⅱ was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-Ⅰ, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical character-istics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-Ⅱ displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-Ⅱ is inhib-ited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The N-terminal sequence (LPSFVDWRQKGVVFPIRNQGQ CGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-α-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-α-CBZ-L-Gln-p-nitrophenyl ester as substrate: Km=0.1634 mM, kcat=121.48 s-1, and kcat/Km =7.4×105 s-1/mM.