The two-hybrid assay was described in 1989 as a method to determine protein-protein interactions in living cells (1 ). The principle of the assay relies on the fact that the DNA binding and transactivation domains are separable and can operate in heterologous contexts in most transcription factors (2 ). The two-hybrid assay exploits this fact by detecting the interaction between two chimeric proteins, each containing one part (either the DNA-binding domain or the transactivation domain) of a transcription factor. A protein-protein interaction between the other parts of the chimeras will bring the two parts of the transcription factor together, producing an increase in transcription from a reporter gene. The first chimera is composed of a DNA binding domain that recognizes the DNA elements in the promoter to be used and one interaction target protein. The yeast activator proteins GAL4 and Lex A have been commonly used as sources of these domains. The second chimera is a fusion of the second interaction target protein with a strong activation domain (for example, the herpes simplex virus activator protein VP16). A complete transactivator protein will be restored only if the two test proteins interact: the GAL4 DNA binding domain of the fusion protein will specifically bind to the multimerized GAL4 binding sites in the luciferase reporter, but cannot activate unless this fusion protein brings the VP16 activation domain into the vicinity of the promoter through a protein-protein interaction between the chimeric proteins (see Fig. 1 ).