Polyphenol oxidases (PPOs) were isolated from cell suspensions of two cultivars of cotton (Gossypium hirsutum L.), and their biochemical characteristics were studied. PPO from Coker 312, an embryogenic cultivar, showed a highest affinity to catechol 20 mM, and PPO from R405-2000, a nonembryogenic cultivar, showed a highest affinity to 4-methylcatechol 20 mM. The optimal pH for PPO activity was 7.0 and 6.0 for Coker 312 and R405-2000, respectively. The enzyme had an optimal temperature of 25 A degrees C and was relatively stable at 20-30 A degrees C. Reducing sodium metabisulfite, ascorbic acid, dithiothreitol, SnCl2, and FeCl3 markedly inhibited PPO activity, whereas its activity was highly enhanced by Mg2+, Ca2+, and Mn2+ and was moderately inhibited by Ba2+, Cu2+, and Zn2+. The analysis revealed a single band on the sodium dodecyl sulfate polyacrylamide gel electrophoresis which corresponded to a molecular weight of 55 kDa for Coker 312 and 42 kDa for R405-2000.