The root nodules of both dormant and non-dormant plants of Alnus glutinosa (L.) Gaertn. were found (by the soybean callus bioassay) to contain levels of cytokinin activity greatly exceeding those of other parts of young vegetative plants. A large, transient increase in cytokinin activity occurred in the nodules at the time of bud break. Similar, although much smaller, increases were detected also in roots and buds. The increase in the level of nodule cytokinin activity was observed both in mature trees and in young pot-grown plants in two successive years. A second peak of cytokinin activity, considered to be derived from cytokinin nucleotides, was found in the nodules of mature trees in midsummer. Analysis of cytokinin extracts of different plant parts by means of a Sephadex LH20 column revealed the presence of three main peaks of activity, with elution volumes corresponding to those of zeatin-9-glucoside, zeatin riboside, and zeatin. While the glucoside-like peak -was predominant in the nodules and leaves it was not detected in root pressure sap. A zeatin ribo-side-like peak was the major cytokinin in the roots and root pressure sap. These findings are discussed in relation to current hypotheses concerning the production, distribution, and possible physiological roles of the cytokinins.