AbstractPolyphenoloxidase (PPO) from red grape cultivar, DeChaunac, grown in New York State was isolated and purified 17‐fold by using Phenyl Sepharose CL‐4B column. Disc gel electrophoresis revealed near homogeneity of three isoenzyme bands. The molecular weight of this enzyme ranged from 73 000 to 85 000. The temperature and pH optima of the purified enzyme were 20 °C and 6.0, respectively. Kinetic studies showed that the thermal inactivation of the PPO followed first‐order kinetics, with the activation energy, Ea = 52.39 Kcal mol−1. The substrate specificity showed a high degree of PPO activity toward o‐diphenolic compounds with the highest affinity toward caffeic acid among substrates studied. The apparent Km values for caffeic acid and 4‐methylcatechol as substrates for the Dechaunac PPO were 16 mmol and 25 mmol, respectively. The most potent inhibitors of the PPO were D.L‐dithiothreitol and sodium metasulphite at the concentration level of 0.5 mmol.