The large serine recombinase, TnpX, from the Clostridium perfringens integrative mobilizable element Tn4451, consists of three domains and has two known DNA binding regions. In this study random and site-directed mutagenesis was used to identify other regions of TnpX that were required for biological activity. Genetic and biochemical analysis of these mutants led to the identification of important TnpX residues in the N-terminal catalytic pocket. In addition, another region of TnpX (aa 243-261), which is conserved within large serine recombinases, was shown to be essential for both excision and insertion. Mutation of charged residues within this region led to a loss of biological activity and aberrant DNA binding. This phenotype was mediated by interaction with the distal DNA binding region (aa 598-707). In these mutants, removal of residues 598-707 resulted in loss of DNA binding, despite the presence of the primary DNA binding region (aa 533-583). Analysis of mutations within the aa 243-261 region indicated that different protein conformations were involved in the insertion and the excision reactions. In summary, we have shown that TnpX is a complex protein that has multiple intra- and intermolecular interaction sites, providing insight into the structural and functional complexity of this important enzyme family.