A series of 9-substituted derivatives of four common naturally occurring cytokinins was tested for growth response in the soybean callus assay. The 9-substituents, 2-carboxyethyl (-C2CO2H), 2-carbo-t-butoxyethyl (-C2CO2 tBu), and 2-nitriloethyl (-C2CN), all reduced the biological activity of the parent compound. The order of activities was, in general,-C2CO2H > -C2CO2 tBu > -C2CN and followed the unsubstituted cytokinin response pattern of E-zeatin (E-Z) > Z-zeatin (Z-Z) > ≅ (R,S)-dihydrozeatin [(diH)Z] >N-(3-methyl-2-butenyl)-1H-purin-6-amine (iP). The nature of the dose-response curves indicate that the activities of the derivatives are a function of their affinities to a common receptor site and that their steric and polar properties determine that affinity.