The roles of three starch branching enzyme isoforms, BE1, BE2 and BE3, in Arabidopsis leaf starch metabolism were investigated by AtBE mutant analysis. We also determined the expression patterns of BEs in Arabidopsis by GUS staining and microarray experiments. The possible correlation of BE gene expression with other genes in the Arabidopsis genome was analyzed to identify putative genes that are functionally correlated with BEs. Motifs in the promoters of the Arabidopsis BE genes were identified to better understand their expression differences. BE2 and BE3 expression levels during a diurnal cycle were evaluated with isoform specific antibodies. Meantime, the in vitro biochemical activities of purified recombinant BE2 and BE3 proteins were investigated by three different assays to better understand their specific functions in starch biosynthesis. Analyses of the structures of the final products from the in vitro BE-amylose reactions were done by FACE with two different forms of amylose.