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Populus euphratica Oliv., a species of the model woody plant genus Populus, is well known for its tolerance to salinity stress, the underlying mechanism of which is a research hotspot. Transient expression of fluorescent fusion proteins is commonly used for rapid assessment of gene functions and interactions, and thus would be useful to study the genes involved in salt tolerance in this species. Our transient gene expression protocol for P. euphratica included a simple protoplast preparation and transformation procedure from suspension cultured cells. The highest protoplast yield (8 x 10(7) g(-1) fresh weight) with high viability (above 90 %) was obtained using an optimized enzyme mix of 4 % (w/v) cellulase R10, 0.5 % (w/v) pectinase, and 0.2 % (w/v) hemicellulase. Factors affecting protoplast transformation efficiency were also optimized: 20 mu g plasmid DNA versus 10(5) protoplasts, and a transformation time of 20 min using PEG, which resulted in a transformation efficiency greater than 50 %. A pair of known markers was simultaneously and correctly expressed in the same P. euphratica protoplasts by co-transformation. The isolation and transformation protocol took 5 h, and results could be obtained within 24 h. This protoplast transient expression system is suitable for studying gene expression, protein localization, and protein-protein interactions in woody plants. In addition, it would be particularly useful for studying the signaling pathway involved in the salt tolerance of P. euphratica in a homologous system, which may not even be possible using protoplasts prepared from other species.