Chymopapain (EC 3.4.22.6) was purified from commercially available dried latex of papaya (Carica papaya) by extraction at acidic pH, cation-exchange chromatography and active site-directed affinity chromatography on immobilized alanyl-phenyl-alaninaldehyde semicarbazone, with elution by mercuric chloride. The product was found by immunoassay to be essentially free of the other cysteine proteinases from papaya, including papaya proteinase IV, and was fully active. The rate of alkylation of the active site cysteine of chymopapain by iodoacetate was found to be sufficiently rapid and selective for this reagent to be used as an active-site titrant.