To understand the roles of the 5'-flanking region of the recognition sites in binding specificity and the affinity of integration host factor (IHF), a variety of DNA fragments with a 13-bp consensus sequence, 5'-WATCAAN4TTR-3'[Friedman, Cell, 55, 545 (1988)], but with different 5'-flanking sequences were investigated by gel retardation and methylation interference assays. It has been well-established that the putative A/T rich element distal from the 5'-end of the consensus made a significant contributions to the binding of IHF. However, many of the DNA fragments used here revealed specific binding to IHF without such an A/T element. Several bases neighboring to the 5'-end of the consensus sequence had significant effects on the binding specificity as well as its affinity, and these results indicate that the sequence-directed bendability of the flanking region plays an important role in the specific recognition by IHF.