The genetic manipulation of Agrobacterium tumefaciens is used to facilitate studies of bacterial gene functions or as a first step in introducing genetic material into transformable plant cells through the use of T-DNA binary vectors. Three methods are commonly used. Transformation with purified plasmid can be done with either electroporation or a simple freeze/thaw transformation method. Alternatively, a mobilizable plasmid can be placed into Agrobacterium using the triparental mating method. Here we present three detailed protocols for Agrobacterium strain construction using electroporation, the freeze/thaw method of transformation, and triparental mating.