Agrobacterium rhizogenes induces growth of aberrant roots with bacterial T-DNA; some roots can spontaneously regenerate into plants carrying T-DNA fragments. T-DNAs contain genes that are expressed in plants which normally lead to root growth and to the synthesis of novel compounds, called opines, used by the agrobacterium. Interestingly, tobacco has been found to contain such T-DNA and is therefore derived from an ancient Agrobacterium infection event. Few examples are known of plants with T-DNA, another one being Linaria vulgaris. Up to now, only a small part of the tobacco T-DNA was identified. Taking advantage of the deep sequencing technique, we sequenced the DNA of one of the two immediate ancestors of tobacco, Nicotiana tomentosiformis, and identified 4 T-DNA sequences: TA, TB, TC and TD, derived from four transformation events. These events could have taken place successively or in parallel. In the latter case, the T-DNAs could have been combined by crosses between different transformants. Each of the four T-DNAs consists of partially repeated sequences. The DNA sequences of these repeats were identical at the time of their introduction into the ancestors, but subsequently diverged. Since divergence is proportional to time this allowed us to reconstruct the order of T-DNA introduction. In tobacco, the TC region is absent and may have been lost. So far, we do not know whether and how the T-DNA genes modify the morphogenic or physiological properties of the plants that contain them. We found that most of the 41 potential T-DNA genes lost their function although one (mas2’) was shown to encode production of the opine desoxyfructosylglutamine. Some tobacco cultivars have a high mas2’ expression, suggesting that mas2’ plays a role in the physiology of these cultivars. Further studies are required to establish the structural and functional basis for the differences in mas2’ expression.