Cytokinins are a class of phytohormones that play a critical role in plant growth and development. sob5-D, an activation-tagging mutant, shows phenotypes typical of transgenic plants expressing the Agrobacterium tumefaciens isopentenyltransferase (ipt) gene that encodes the enzyme catalyzing the first step of cytokinin biosynthesis. The sob5-D mutant phenotypes are caused by over-expression of a novel gene, SOB5. Sequence analysis places SOB5 in a previously uncharacterized family of plant-specific proteins. A translational fusion between SOB5 and the green fluorescent protein reporter was localized in the cytoplasm as well as associated with the plasma membrane when transiently expressed in onion epidermal cells. Analysis of transgenic plants harboring an SOB5:SOB5-beta-glucuronidase (GUS) translational fusion under the control of the SOB5 promoter region showed GUS activity in vegetative tissues (hydathodes and trichomes of leaves, shoot meristems and roots) as well as in floral tissues (pistil tips, developing anthers and sepal vasculature). Cytokinin quantification analysis revealed that adult sob5-D plants accumulated higher levels of trans-zeatin riboside, trans-zeatin riboside monophosphate and isopentenyladenine 9-glucoside when compared to the wild-type. Consistent with this result, AtIPT3 and AtIPT7 were found to be up-regulated in a tissue-specific manner in sob5-D mutants. Physiological analysis of the sob5-D mutant demonstrated reduced responsiveness to exogenous cytokinin in both root-elongation and callus-formation assays. Taken together, our data suggest a role for the novel gene SOB5 in cytokinin-mediated plant development.