Isoprenoids comprise a broad range of natural products that are synthesized by the condensation of isopentenyl diphosphate and dimethylallyl diphosphate. Plants have two distinct routes for the synthesis of these common isoprenoid precursors, the methylerythritol phosphate (MEP) pathway in plastids and the mevalonate (MVA) pathway in the cytosol. We have extensively studied the role of MEP and MVA pathways in the biosynthesis of gibberellins (GAs) and cytokinins (CKs) in plants. To determine the contribution of MEP and MVA pathways to hormone biosynthesis, we performed tracer experiments in which each metabolite from the MEP or MVA pathways are selectively and efficiently ^<13>C-labeled in Arabidopsis. We found that GAs are mainly produced from the MEP pathway in plastids, but also partially from the MVA pathway in the cytosol. We also showed that a large fraction of prenyl side chains of isopentenyladenine-type and trans-zeatin-type CKs are mainly produced from the MEP pathway, while those of cis-zeatin type CKs are predominantly derived from the MVA pathway. Moreover, we investigated the biosynthesis of CKs in crown gall, which is induced by infection of Agrobacterium tumefaciens to host plants. Our study showed that Agrobacterium efficiently produces trans-zeatin-type CKs by creating a non-plant-type CK biosynthetic pathway in plastids. This result provided new insight into the function of Agrobacterium CK biosynthetic enzyme that can move into plastids without typical signal peptides.