Xanthomonas axonopodis pv. citri (Xac) is the causative agent of the citrus canker, an important disease in orange trees. The genome of Xac has two type 4 secretion systems (T4SS), one chromosomal- and one plasmid-encoded. T4SS is important for virulence and survival of different bacterias, including the phytopathogen Agrobacterium tumefaciens and the human pathogens Legionella pneumophila, Helicobacter pylori and Bordetella pertussis. Our group previously identified new protein-protein interactions among Xac T4SS subunits by yeast two-hybrid assays. One interaction network (VirD4-Xac2609-Xac2610-VirB11) that we identified involves two ATPases (VirD4 and VirB11) and two hypothetical proteins (Xac2609 and Xac2610) coded by the chromosomal vir locus. In this work we characterize the Xac2609-2610 interaction in greater detail using purified proteins. The genes encoding the proteins Xac2609 and Xac2610 were cloned in the pET11a and the pET28a vectors, respectively. The Xac2610 has a potential N-terminal transmembrane region, and we cloned the gene with (Xac2610_1-267) and without (Xac2610_22-267) the first 21 residues, with and without a N-terminal His-tag. The Xac2610_22-267 without the transmembrane domain is soluble while the full-length protein is insoluble. The proteins were expressed in E. coli BL21(DE3)RP cells and both were purified using standard chromatographic methods. The purifications were analyzed by SDS-PAGE and MALDI-TOF mass spectrometry. The Xac2609-2610 interaction was confirmed by “pull down”, limited proteolysis, fluorescence, circular dichroism and thermal denaturation assays. Limited proteolysis with trypsin and chymotrypsin revealed an increased stability of specific proteolytic fragments in the Xac2609-2610 complex. Fluorescence spectroscopy showed that the Xac2609-2610 interaction has a high affinity, with a Kd < 10^-9 M. Thermal denaturation studies (fluorescence and circular dichroism) indicated that Xac2610 stabilizes and raises the Tm of Xac2609. The VirD4-Xac2609-Xac2610-VirB11 interaction network is possibly important for the chromosomal T4SS function and Xac2609 may be a T4SS substrate. Furthermore, Xac2610 has a potential nuclear localization signal (PKKK) and may act in the plant nucleus. In the future, we plan to study VirD4-2609 and VirB11-2610 interactions and the cellular localization of Xac2609 and Xac2610.