Peroxidase in tobacco callus tissue (Nicotiana tabacum, cv. White Gold) was resolved into three groups of isoenzymes by polyacrylamide gel electrophoresis, and a combined action of cytokinin, auxin, and gibberellin in their formation was clearly demonstrated. The most significant change was in a group of fast-migrating isoperoxidases, the development of which required both kinetin and indoleacetic acid. Kinetin was most stimulatory at 0.2 μM but became inhibitory with increasing concentrations. Indole acetic acid was effective at concentrations from 0.1 to 100 μM with an optimum at 10 μM. With both kinetin and indoleacetic acid at optimal concentrations, addition of gibberellic acid further increased the contents of the fast migrating isoperoxidases, but it was inactive in the absence of indoleacetic acid or in the presence of kinetin in 5 μM or higher concentrations. Cycloheximide, actinomycin D, and abscisic acid inhibited the formation of the fast-migrating peroxidases. Formation of the fast-migrating isoperoxidase in the tissue was associated with tumor-type growth.2,4-Dichlorophenoxyacetic acid had a dual effect on peroxidase; at low concentrations (0.1 to 1 μM) it promoted a fast-migrating isoperoxidase; at high concentrations (10 to 100 μM) it inhibited the fast-migrating isoperoxidase but caused a significant increase in other isoperoxidases of lower electrophoretic mobilities.