Cytokinins (CKs) are phytohormones that play an important role in plant growth and development. Although the first naturally produced CK, zeatin, was isolated almost four decades ago, no endogenous gene has been shown to produce active CKs in planta. In an activation tagging experiment we have identified a petunia line that showed CK-specific effects including enhanced shooting, reduced apical dominance and delayed senescence and flowering. This phenotype correlated with the enhanced expression of a gene we labelled Sho (Shooting). Sho, which encodes a protein with homology to isopentenyl transferases (IPTs), also causes CK-specific effects when expressed in other plant species. In contrast to the ipt gene from Agrobacterium, which primarily increases zeatin levels, Sho expression in petunia and tobacco especially enhances the levels of certain N6-(delta2-isopentenyl) adenosine (2iP) derivatives. Our data suggest that Sho encodes a plant enzyme whose activity is sufficient to produce active CKs in plants.