AbstractAgrobacterium species are plant‐associated relatives of the rhizobia. Several species cause plant diseases such as crown gall and hairy root, although there are also avirulent species. A. tumefaciens is the most intensively studied species and causes crown gall, a neoplastic disease that occurs on a variety of plants. Virulence is specified by large plasmids, and in the case of A. tumefaciens this is called the Ti (tumor‐inducing) plasmid. During pathogenesis virulent agrobacteria copy a segment of the Ti plasmid and transfer it to the plant, where it subsequently integrates into the plant genome, and expresses genes that result in the disease symptoms. A. tumefaciens has been used extensively as a plant genetic engineering tool and is also a model microorganism that has been well studied for host‐microbe associations, horizontal gene transfer, cell‐cell communication, and biofilm formation. This unit describes standard protocols for simple phenotypic characterizations of A. tumefaciens. Curr. Protoc. Microbiol. 25:3D.3.1‐3D.3.14. © 2012 by John Wiley & Sons, Inc.