Reversible inversion of the DNA element fimS is responsible for the phase variable expression of type 1 fimbriae in Escherichia coli. The FimB tyrosine integrase site-specific recombinase inverts fimS in the on-to-off and off-to-on directions with approximately equal efficiencies. However, when DNA supercoiling is relaxed, fimS adopts predominantly the on orientation. This orientational bias is known to require binding of the nucleoid-associated protein LRP within fimS. Here we show that binding of the IHF protein to a site immediately adjacent to fimS is also required for phase-on orientational bias. In the absence of both LRP and IHF binding, fimS adopts the off orientation and the H-NS protein is required to maintain this alternative orientational bias. Thus, fimS has three Recombination Directionality Factors, H-NS, IHF and LRP. The relevant H-NS binding site straddles the left inverted repeat in phase-off fimS and this site is disrupted when fimS inverts to the on orientation. The inversion of fimS with the associated creation and removal of an H-NS binding site required for DNA inversion biasing represents a novel mechanism for modulating the interaction of H-NS with a DNA target and for influencing a site-specific recombination reaction.