Gene overexpression as a means to determine plant gene function has been used almost since the first plant transformation protocols became viable. The goal of these experiments, as in classical genetic experiments, is to observe any phenotypic change associated with changing the expression of a gene of interest—in this case overexpression. Any phenotypic changes are interpreted, and the native gene’s function is deduced based on the pathways or biochemistries that are altered in the transformants. Overexpression experiments may be particularly suitable in instances when genes are functionally redundant, when a plant species does not have good genetics, or when a knockout mutation is particularly deleterious. This chapter is intended as a general protocol for producing gene overexpression constructs, starting with genomic DNA, RNA, or an isolated clone, for use in plants that are transformable by Agrobacterium .