The recombinational enhancer of the Hin inversion system in Salmonella stimulates recombination in vitro 150-fold in the presence of the Escherichia coli host factor Fis. To gain an understanding of the roles of the enhancer and Fis in stimulating the Hin-mediated inversion reaction, we have used nuclease and chemical protection/interference studies and gel retardation assays to examine the interactions between Fis and the recombinational enhancer. These studies combined with mutational analysis defined the enhancer sequences required for Fis binding and function. Fis binds with different affinities to two domains within the enhancer sequence. The binding of Fis at each domain is independent of the occupancy of the other domain and appears to be to opposite faces of the DNA helix. These results support a model for the role of the recombinational enhancer in Hin-mediated inversion in which the interaction between Hin bound at recombination sites and Fis bound to each domain of the recombinational enhancer results in a structure with the proper alignment and topology to promote DNA inversion.