Abstract: A method and a kit for detecting interactions between three or more proteins, in vivo, using reconstitution of the activity of a transcriptional activator is provided. Reconstitution of the transcriptional activator makes use of chimeric genes which express hybrid proteins. In one embodiment, three types of hybrid proteins are prepared. The first hybrid contains the DNA-binding domain of a transcriptional activator fused to the first test protein. The second hybrid protein contains a transcriptional activation domain fused to the second test protein. The third hybrid protein contains a nuclear localization peptide fused to a third test protein and mediates assembly of the three-protein complex involving the three hybrids. If the three test proteins are able to interact, they bring into close proximity the two domains of the transcriptional activator.