Transposase activity that mediates the mobility of class II transposable elements, is most commonly initiated by the assembly of higher order synaptic complexes, called transpososomes. The formation of these complexes, that contain the transposable element's DNA as well as two or more molecules of the transposase, is dependent on interactions between transposase molecules. Using the yeast Two-Hybrid system, we were able to identify three regions mediating multimerization of the Hermes transposase, an element used for germline transformation of insects belonging to the hAT family of transposable elements. One region facilitating protein binding of Hermes transposase molecules was found within the first 252 amino acids of the transposase. The second region was located at the C-terminus of the transposase, and was found to be specific for Hermes transposase multimerization. Amino acids 551-569 were not only required for multimerization but were also necessary for transposition of the element. The third region was located between amino acids 253 and 380 and was found to eliminate the non-specific protein binding ability of the N-terminal protein interaction region but was required for the specific protein binding ability of the C-terminal region of the transposase. Five point mutations affecting the structural integrity of the C-terminal multimerization region abolished or significantly reduced transpositional activity. The same region had been previously identified to mediate dimerization in Activator (Ac), another hAT element, indicating that hAT transposase multimerization is likely to be a prerequisite for mobility of their elements.