Proteolytic activity present in the latex of Asclepias syriaca L. has been isolated and purified. High speed centrifugation was used to separate the gums and resins of fresh latex from an aqueous serum layer. The serum layer (pH 5.2) contains 3.7% (w/v) protein and all the proteolytic activity present in fresh latex. This serum layer was freeze-dried to yield a dry soluble powder retaining full enzymatic activity. The activity was isolated and separated into 2 fractions, termed A and B (representing 32% of the soluble protein), on an organomercurial-agarose affinity column. A and B were further resolved into 10 active fractions, termed Al to A5 and Bl to B5, using CM-sepharose CL-6B ion-exchange chromatography. These multiple enzymes did not arise in the purification procedure and appear to exist in vivo. Two of these fractions, A3 and B5, have been purified to homogeneity using re-chrornatography on CM-sepharose CL-6B, Homogeneity was confirmed by polyacrylamide gel electrophoresis (±SDS), ion-exchange chromatography and gel-filtration chromatography. The yield of A3 and B5 was 8 mg and 9 mg respectively from 1 g of crude powder (~1Q ml of fresh latex). Both enzymes were active towards ester, amide and protein substrates and showed autodigestive activity under strongly reducing conditions They were activated by sulfhydryl-containing reducing agents and strongly inhibited by the sulfhydryl reagents iodoacetic acid, p-chloromercurihenzoate and sodium tetrathionate. The optimum pH for the digestion of casein was 7,5 to 8.5 for A3 and 7.Q to 7.5 for B5. Both A3 and B5 behaved as single polypeptides in SDS gel electrophoresis, each giving a molecular weight of 23,000. In sephadex G-1QQ gel-fi1tration chromatography, both enzymes eluted as single proteins, A3 showing a molecular weight of 24,000 and B5 a molecular weight of 19,000. Both these enzymes were concluded to be sulfhydryl proteases. Several possible explanations for the multiple nature of these proteases, their functions and the role(s) of latex in Asalepias syriaca L. are suggested. Future research possibilities are discussed.