Agrobacterium-mediated transformation of rice is now used in many laboratories worldwide. Several protocols have been developed and fine-tuned for particular genotypes, including commercial genotypes, making use of either mature seeds or immature embryos as target tissue for Agrobacterium infection. In this chapter, we describe a rapid and user-friendly protocol based on mature seeds that can deliver transgenic rice plant-lets within 2 mo. The protocol described is based on the use of the nptII selectable marker gene. The tissue culture steps rely on Agrobacterium infection of whole young seedlings and on the induction and proliferation of highly embryogenic tissue. Importantly, we have validated the robustness and reliability of this protocol at a high-throughput scale for several japonica genotypes. We also provide some key features that can be further explored for the fine-tuning of this transformation protocol for any other genotype with a particular emphasis on the importance of tissue handling and subculture sequence.