The transfer of plasmid DNA molecules between bacterial cells is achieved by a large array of conjugative transfer proteins which assemble into both cytoplasmic and membrane-associated complexes. TrhC is a membrane-associated protein that is required for the transfer of the IncHI1 resistance plasmid R27. Homologous proteins are encoded in all known conjugative systems, and each contains characteristic nucleoside triphosphate (NTP)-binding domains. An assembly of R27-encoded proteins was previously visualized by use of a TrhC-green fluorescent protein fusion, which appeared as discrete membrane-associated fluorescent foci. We have utilized this experimental system to determine the requirements for assembly of this TrhC-associated protein complex, and we found that 12 of the other 18 R27 transfer proteins are required for focus formation. An individual focus possibly represents a subassembly comprised of some or all of these transfer proteins. These data support the notion that the transfer apparatus is a multicomponent structure. In contrast, substitutions and deletions within TrhC NTP-binding motifs had minor effects on focus formation, but these mutations did affect plasmid transfer and bacteriophage susceptibility. These results indicate that TrhC requires intact NTP-binding motifs to function during conjugative transfer but that these motifs are not essential for the assembly of TrhC into a complex with other transfer proteins.