E. coli integration host factor (IHF) both participates directly in phage lambda site-specific recombination and regulates the expression of phage and bacterial genes. Using protection from nuclease and chemical attack as an assay, we examined the interaction of IHF with DNA. We found that IHF is a specific DNA binding protein that interacts with three distinct segments of attP, the recombination site carried by phage lambda. We also found that specific IHF binding sites are located in non-att DNA. Several non-att IHF binding sites that we have identified are adjacent to genes whose expression is altered in IHF mutants. From comparison of the sequences protected by IHF, we suggest that the critical determinant in specific IHF-DNA interaction is contained in the sequence T.PyAA...PuTTGaT.A.PuTT...PyAACtA.