Type IV secretory systems are a group of bacterial transporters responsible for the transport of proteins and nucleic acids directly into recipient cells. Such systems play key roles in the virulence of some pathogenic organisms and in conjugation-mediated horizontal gene transfer. Many type IV systems require conserved "coupling proteins," transmembrane polypeptides that are critical for transporting secreted substrates across the cytoplasmic membrane of the bacterium. In vitro evidence suggests that the functional form of coupling proteins is a homohexameric, ring-shaped complex. Using a library of tagged mutants, we investigated the structural and functional organization of the F plasmid conjugative coupling protein TraD by coimmunoprecipitation, cross-linking, and genetic means. We present direct evidence that coupling proteins form stable oligomeric complexes in the membranes of bacteria and that the formation of some of these complexes requires other F-encoded functions. Our data also show that different regions of TraD play distinct roles in the oligomerization process. We postulate a model for in vivo oligomerization and discuss the probable participation of individual domains of TraD in each step.