The vast majority of proteins functions in complex with one or more of the same or other proteins, indicating that protein-protein interactions play crucial roles in biology. Here, we present a beta-galactosidase reconstitution-based bacterial two-hybrid system in which two proteins of interest are fused to two non-functional but complementing beta-galactosidase truncations (Delta alpha and Delta omega). The level of complemented beta-galactosidase activity, driven by the protein-protein recognition between both non-beta-galactosidase parts of the chimeras, reflects whether or not the proteins of interest interact. Our approach was validated by reconfirming some well-established Escherichia coli cytoplasmic and membranous interactions, including well-chosen mutants, and providing the first in vivo evidence for the transient periplasmic interaction between Rhodobacter capsulatus cytochrome c2 and cytochrome c peroxidase. We demonstrated the major advantages of this in vivo two-hybrid technique: i) analyses of interactions are not limited to particular cellular compartments, ii) the potential of using the system in mutation-driven structure-function studies, and iii) the possibility of its application to transiently interacting proteins. These benefits demonstrate the relevance of the method as a powerful new tool in the broad spectrum of interaction assessment methods.