AbstractCytokinins (CKs), a class of phytohormones with vital roles in growth and development, occur naturally with various side-chain structures, includingN6-(Δ2-isopentenyl)adenine-,cis-zeatin- andtrans-zeatin (tZ)-types. Recent studies in the model dicot plant Arabidopsis (Arabidopsis thaliana) have demonstrated that tZ-type CKs are biosynthesized via cytochrome P450 monooxygenase (P450) CYP735A and have a specific function in shoot growth promotion. Although the function of some of these CKs has been demonstrated in a few dicotyledonous plant species, the importance of these variations and their biosynthetic mechanism and function in monocots and in plants with distinctive side-chain profiles other than Arabidopsis, such as rice (Oryza sativa), remain elusive. In this study, we characterizedCYP735A3andCYP735A4to investigate the role of tZ-type CKs in rice. Complementation test of the Arabidopsis CYP735A-deficient mutant and CK profiling of loss-of-function rice mutantcyp735a3 cyp735a4demonstrated thatCYP735A3andCYP735A4encode P450s required for tZ-type side-chain modification in rice.CYP735Asare expressed in both roots and shoots. Thecyp735a3 cyp735a4mutants exhibited growth retardation concomitant with reduction in CK activity in both roots and shoots, indicating that tZ-type CKs function in growth promotion of both organs. Expression analysis revealed that tZ-type CK biosynthesis is negatively regulated by auxin, abscisic acid, and CK and positively by dual nitrogen nutrient signals, namely glutamine-related and nitrate-specific signals. These results suggest that tZ-type CKs control the growth of both roots and shoots in response to internal and environmental cues in rice.