Agrobacterium tumefaciens can genetically transform eukaryotic cells. In many bacteria, pili are required for interbacterial DNA transfer. The formation of pili by Agrobacterium required induction of tumor-inducing (Ti) plasmid-encoded virulence genes and growth at low temperature. A genetic analysis demonstrated that virA, virG, virB1 through virB11, and virD4 are the only Ti plasmid genes necessary for pilus assembly. The loss and gain of pili in various mutants correlated with the loss and gain of transferred DNA (T-DNA) transfer functions, which is consistent with the view that Agrobacterium pili are required for transfer of DNA to plant cells in a process similar to that of conjugation.