The bacterial adenylate cyclase two-hybrid (BACTH) system has been widely used to characterize protein–protein interactions in the prokaryotic world. This system relies on the interaction-mediated reconstitution of adenylate cyclase activity inEscherichia coliby bringing together two complementary fragments of the catalytic domain of the adenylate cyclase toxin ofBordetella pertussis. A limiting factor in performing large-scale two-hybrid interaction screens with full-length open reading frames (ORFs) is the need to clone each ORF individually into the plasmids used to express the hybrid proteins. The Gateway® (GW) cloning system (Life Technologies, Grand Island, NY, USA) partially circumvents this limitation, and we describe here modifications to the BACTH system for compatibility with this recombineering technology. We validated and tested the functionality of the BACTH Gateway (BACTHGW) system using several models of protein–protein interactions, focusing particularly on those involved in bacterial cell division. We further modified the BACTH plasmids to incorporate a transmembrane (TM) segment downstream of the cyclase fragments to permit analysis of extracytoplasmic protein interactions. This approach was also useful to identify putative TM segments and to experimentally validate bioinformatically identified TM domains. The BACTHGW system will prove a useful addition to the study of protein–protein interactions.