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Cleavage and polyadenylation of precursor messenger RNA (pre-mRNA) is an essential processing event during mRNA maturation. Most of the understanding of the protein factors required for this process comes from yeast and mammals in which successful in vitro cleavage and polyadenylation systems have been extensively employed. In contrast, limited information about plant polyadenylation factors and their functions has been largely from bioinformatics and genetics studies more recently, due to the lack of a plant in vitro system. Using tandem affinity purification (TAP) methods, plant cleavage and polyadenylation specificity factor (CPSF) was isolated and identified from Arabidopsis thaliana. By compiling the in vivo interaction data from TAP purification, as well as available yeast two-hybrid assay data from literature, a plant CPSF model is proposed. According to this model, the Arabidopsis CPSF possesses AtCPSF30, AtCPSF73-I, AtCPSF73-II, AtCPSF100, AtCPSF160, AtFY, and AtFIPS5. Among them, AtCPSF100 serves as a core on which other factors are associated, except for AtFIPS5. AtFIPS5 associates to the CPSF complex indirectly by interacting with AtCPSF30. Protein factors from other complexes, such as AtSYM5, AtCLPS3 and AtPCFS4, may associate with CPSF through AtCPSF30 directly or indirectly. These results show that plant CPSF shares many similarities with its yeast and mammalian counterparts, despite some obvious distinct features. To reconstitute an in vitro assay system for plant mRNA 3'-end processing, protein extracts from Arabidopsis suspension cell cultures over-expressing some polyadenylation-related protein factors, such as AtCPSF30, AtCPSF73-I, AtCPSF73-II, and AtCLPS3, were successfully used in regenerating the cleavage reaction. The characteristics of the plant in vitro cleavage system were further studied by using one of these over-expressed factors, AtCPSF73-I. This in vitro cleavage system has the expected features: cleaves the substrate RNA at the same locations as when processed in vivo; the cleavage activity is dependent on the polyadenylation signals; and the cleavage leaves a 3'-hydroxyl group at the 3'-end for polyadenylation. Analysis of the system also showed the potential involvement of both endonuclease and 3'-5'exonuclease activities. An endo-exonuclease dualism model was proposed for pre-mRNA cleavage in plants. This pioneering system will serve as a platform to further explore plant mRNA 3'-end formation mechanisms and other RNA processing events.