Isolated protoplasts serve as a transient expression system that is highly representative of stable transgenics in terms of transcriptome responses. They can also be used as a cellular system to study gene transactivation and nucleocytoplasmic protein trafficking. They are particularly useful for systems studies in which stable transgenics and mutants are unavailable. We present a protocol for the isolation and transfection of protoplasts from wood-forming tissue, the stem-differentiating xylem (SDX), in the model woody plant Populus trichocarpa. The method involves tissue preparation, digestion of SDX cell walls, protoplast isolation and DNA transfection. Our approach is markedly faster and provides better yields than previous protocols; small (milligrams)- to large (20 g)-scale SDX preparations can be achieved in ~60 s, with isolation of protoplasts and their subsequent transfection taking ~50 min. Up to ten different samples can be processed simultaneously in this time scale. Our protocol gives a high yield (~2.5 × 10(7) protoplasts per g of SDX) of protoplasts sharing 96% transcriptome identity with intact SDX.