This paper describes a bacterial two-hybrid system that allows the in vivo screening and selection of functional protein-protein interactions. The system is based on the reconstitution of a signal transduction pathway in an Escherichia coli cya strain, utilizing the positive control exerted by cAMP. Two interacting proteins are genetically fused to complementary fragments (T25 and T18) of the catalytic domain of Bordetella pertussis adenylate cyclase. When these fragments are brought into close proximity, they functionally complement each other, leading to cAMP synthesis. Cyclic AMP then triggers the transcriptional activation of catabolic operons, such as lactose or maltose, resulting in a characteristic phenotype. This system offers the unique advantage of spatially separating the association of the hybrid proteins from the transcriptional activation readout, allowing versatile screening procedures for ligands binding to a given "bait" or molecules/mutations blocking a given interaction. The authors demonstrate the system's effectiveness by showing that it can detect interactions between small peptides, bacterial proteins, and eukaryotic proteins. The bacterial two-hybrid system provides a powerful tool for identifying and characterizing protein-protein interactions in a simple genetic test.This paper describes a bacterial two-hybrid system that allows the in vivo screening and selection of functional protein-protein interactions. The system is based on the reconstitution of a signal transduction pathway in an Escherichia coli cya strain, utilizing the positive control exerted by cAMP. Two interacting proteins are genetically fused to complementary fragments (T25 and T18) of the catalytic domain of Bordetella pertussis adenylate cyclase. When these fragments are brought into close proximity, they functionally complement each other, leading to cAMP synthesis. Cyclic AMP then triggers the transcriptional activation of catabolic operons, such as lactose or maltose, resulting in a characteristic phenotype. This system offers the unique advantage of spatially separating the association of the hybrid proteins from the transcriptional activation readout, allowing versatile screening procedures for ligands binding to a given "bait" or molecules/mutations blocking a given interaction. The authors demonstrate the system's effectiveness by showing that it can detect interactions between small peptides, bacterial proteins, and eukaryotic proteins. The bacterial two-hybrid system provides a powerful tool for identifying and characterizing protein-protein interactions in a simple genetic test.