Cytokinins from leaf laminae, buds, petioles, stems, roots, and root exudate of mature vegetative plants of Xanthium strumarium L. were extracted, fractionated, and partially characterized by means of column chromatography with Sephadex LH20. Two peaks of cytokinin activity with elution volumes corresponding to zeatin and zeatin riboside were detected, in varying concentrations, in all plant parts. A third cytokinin, detected only in petioles and in expanding and mature leaves, eluted off the Sephadex column before zeatin riboside. This cytokinin (peak ‘a’) was converted to zeatin or to a zeatin-like cytokinin following both acid hydrolysis and treatment with β-glucosidase. Peak ‘a’ was not detected in buds or in the youngest developing leaves but was the predominant cytokinin present in half-expanded and more mature leaves. By contrast, the zeatin riboside-like peak (peak ‘b’) constituted the major cytokinin in root exudate, apical buds, and the youngest developing leaves, while not greatly contributing to the cytokinin content of mature leaves. The detopped root system was shown to be capable of cytokinin production. The distribution of cyrtokinins in the plant is discussed in relation to their probable origin in the root system.