Agrobacterium tumefaciens has been widely used to transform numerous plant species. Frequently, investigators want to place more than one gene into Agrobacterium in order to manipulate various bacterial functions during plant genetic transformation. These genes are frequently brought into the bacterium by multiple plasmids. It is difficult to maintain several plasmids in the same bacterium without the use of a variety of antibiotics. However, the use of some antibiotics is not feasible for certain Agrobacterium strains. Also, too many different antibiotics in the culture medium generally results in slow growth of Agrobacterium. Therefore, it is to one's advantage to place genes of interest onto the bacterial chromosome to eliminate the use of multiple antibiotics during bacterial growth. This chapter describes in detail a method to integrate a gene of interest into the pgl/picA locus of the Agrobacterium tumefaciens C58 chromosome. The integrated gene is stably maintained as single copy per cell without the need for selection.