Plant cell apoplast plays a major role in defensing abiotic stresses such as drought,salinity,and it is composed of cell wall matrix and extracellular spaces.The extracellular proteins(ECPs) are one of main composition in apoplast.Poplar is the pattern of species in tree genomics and proteomics research.In this paper,the methods of extraction,purification and two-dimensional electrophoresis about ECPs were studied by use of leaf and stem segments as sample from NL895 poplar(Populus deltoides×Populus euramericana cv.,NL895).The results were that the effect of cell wall proteins(CWPs) from leaf segments(>10 g) which was extracted for 10 min on ultrasonic processor was better than other methods.By measuring G-6PDH enzyme activity,it was found the rate of cell cytoplasmic contamination was only 2.9% in the CWPs from leaf segments,and was 14.1% in the apoplastic proteins from stem sections.The optimum system for ECPs analysis was leaf segments which purified by method of acetone/precipitation,CaCl₂,24 cm pH 4-7 IPG strips,and loading protein sample of 500 μg.More than 550 protein spots with clear background were observed in 2-D electrophoresisgel.This work preliminarily established the optimum system including extraction,separation,purification and 2-DE electrophoresis technology for poplar ECPs,and this protocol could be beneficial to develop high-level ECPs studies in poplar.