Isozymes of tobacco pith polyphenoloxidases (o-diphenol oxidase, EC 1.10.3.1) were separated electrophoretically from fresh pith of intact plants and from cultured pith sections. Extracts of fresh pith contained a poorly resolved complex of two to three anodic bands after starch gel electrophoresis at alkaline pH. This anodic complex was more active with chlorogenic acid than with 3,4-dihydroxyphenylalanine and was found in greater activity per gram fresh weight of tissue in younger internodes than in older ones. The longitudinal gradient of activity was thus the opposite of that found for the constitutive isozymes of peroxidase.A well defined cathodic band of polyphenoloxidase activity appeared after culture of pith in modified White's medium with shaking. This band, which was more active with 3,4-dihydroxyphenylalanine than with chlorogenic acid, could be detected after 1 to 2 days of incubation. Its appearance was enhanced by the addition of 10 mum indoleacetic acid; kinetin (1 mum tended to prevent this indoleacetic acid effect). Such hormonal control is opposite to that previously reported for the rapidly appearing new isozymes of peroxidase. The pattern of the major isozymes associated with polyphenoloxidase activities differs from that of peroxidase.