ABSTRACT: We have investigated the mechanism of action of 4-hexylresorcinol (4-HR) and ascorbic acid (AA) on the polyphenol oxidase (PPO) catalyzed oxidation of phenolic substrates. Incubation of PPO with 4-HR diminishes strongly PPO activity. This effect can be erroneously interpreted, due to the high affinity of 4-HR for PPO, as irreversible inactivation of PPO. However, PPO activity can be recovered by dialysis after incubation with 4-HR. 4-hexylresorcinol is a canonical enzyme inhibitor that binds preferentially to the oxy form of PPO. It is a mixed-type inhibitor, because it influences both apparent Vmax (1.26 compared with 0.4 units in the absence and presence of 4-HR, respectively) and Km values (0.28 mM compared with 0.97 mM in the absence and in the presence of 4-HR, respectively) of PPO. AA can prevent browning by 2 different mechanisms: In the absence of PPO substrates it inactivates PPO irreversibly, probably through binding to its active site, preferentially in its oxy form. In the presence of PPO substrates, AA reduces PPO oxidized reaction products, which results in a lag phase when measuring PPO activity by monitoring dark product formation but not when monitoring O2 consumption. The simultaneous use of both 4-HR and AA on PPO results in additive prevention of browning.