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Cytokinin-binding protein was isolated from tobacco culture cells and purified by bioaffinity and Sephadex G-200 column chromatographies. The affinity column was prepared by coupling periodate-oxidizing N6-(A2-isopenteny1)adenosine to cyanogen-bromide-activated Sepharose 4B with adipic acid dihydrazide as a spacer arm, followed by borohydride reduction of the oxidized ribose moiety of the cytokinin riboside. The amount of cytokinin molecule coupled to the gel ranged from 6 to 7 pmol/ml gel and the coupling reaction proceeded most efficiently in the p H range of 5-6. The binding capacity of the column was about 380 pg protein/ml wet gel. The amount of binding protein isolated depended upon the age of tissues. Sephadex G-200 chromatographic filtration of the absorbed fraction from the affinity column yielded a glycoprotein and a protein fractions with molecular mass of 123 000 and 8500 A 300 respectively. Results of binding experiments with high and low specific activities of radioactive N6-(A2-isopentenyl)adenine at different concentrations indicate the presence of at least two cytokinin-binding sites in the protein fraction. The high-affinity site is heat labile and has high affinity (apparent & of 8.8 x lo7 M) towards cytokinins. The binding of cytokinin to this site is affected by ionic strength and pH. Tests for binding specificity showed that biologically inactive adenine and cytokinin analogues were ineffective as competitors, whereas biologically active cytokinins competed for the binding of 3H-labeled N6-(A2-isopenteny1)adenine. The order of effectiveness of the cytokinins tested in the competing binding assay follows only partially the reported cytokinin activity order of the tobacco assay. These results suggest that if the binding protein is indeed involved in the cytokinin action in vivo, then the regulatory action of the hormone may be expressed by several mechanisms.Studies of the regulatory action of the plant hormone, cytokinin, have revealed that there may be specific cytokinin binding protein(s) present in various plant tissues [l-61, and the significance of the presence of the protein with regard to the hormone action in higher plants is still not clear. To understand the mechanism of cytokinin action in inducing cell division and differentiation, the isolation and purification of their receptor molecule(s) as well as study of the interaction of the hormone with its presumed binding site(s) should be indispensable. Isolation and partial characterization of specific cytokinin-binding molecule(s) has been achieved from tobacco cells [2,3], Abhreviations. Ade, adenine; i'Adc, Nb-(d2-isopentenyl)adenine; i6Ado, N6-(d2-isopentenyl)adenosine; io' Ade, 6-(4-hydroxy-3-methyl-2-buteny1amino)purine; io'Ado, 6-(4-hydroxy-3-methyI-2-butenylamino)-9-~-ribofuranosylpurine; i6Ado"", N6-(d2-isopenteny1)adenosine oxidized with periodate; i'Ado""-Sepharose gel, i6Ado"" attached to Sepharose-adipic acid dihydrazide; i6Ado""-IedSepharose gel, i6Ado""-Sepharose gel was reduced with NaBH4.wheat germ [1,5,6] and moss...