Cytokinins, which are central regulators of cell division and differentiation in plants, are adenine derivatives carrying an isopentenyl side chain that may be hydroxylated. Plants have two classes of isopentenyltransferases (IPTs) acting on the adenine moiety: ATP/ADP isopentenyltransferases (in Arabidopsis thaliana, AtIPT1, 3, 4–8 ) and tRNA IPTs (in Arabidopsis, AtIPT2 and 9 ). ATP/ADP IPTs are likely to be responsible for the bulk of cytokinin synthesis, whereas it is thought that cis -zeatin ( c Z)-type cytokinins are produced possibly by degradation of cis -hydroxy isopentenyl tRNAs, which are formed by tRNA IPTs. However, these routes are largely hypothetical because of lack of in vivo evidence, because the critical experiment necessary to verify these routes, namely the production and analysis of mutants lacking AtIPT s, has not yet been described. We isolated null mutants for all members of the ATP/ADP IPT and tRNA IPT gene families in Arabidopsis . Notably, our work demonstrates that the atipt1 3 5 7 quadruple mutant possesses severely decreased levels of isopentenyladenine and trans -zeatin ( t Z), and their corresponding ribosides, ribotides, and glucosides, and is retarded in its growth. In contrast, these mutants possessed increased levels of c Z-type cytokinins. The atipt2 9 double mutant, on the other hand, lacked isopentenyl- and cis -hydroxy isopentenyl-tRNA, and c Z-type cytokinins. These results indicate that whereas ATP/ADP IPTs are responsible for the bulk of isopentenyladenine- and t Z-type cytokinin synthesis, tRNA IPTs are required for c Z-type cytokinin production. This work clarifies the long-standing questions of the biosynthetic routes for isopentenyladenine-, t Z-, and c Z-type cytokinin production.