In its natural host, Bacillus thuringiensis, the insertion sequence IS231A is preferentially inserted into the terminal inverted repeats of the transposon Tn4430. Using a novel transposition assay, we demonstrate that the Tn4430 ends behave as insertion hot spots for IS231A in Escherichia coli. Sequence analysis reveals that IS231A insertion sites match the 5'-GGG(N)5CCC-3' consensus. However, this consensus is not the only determinant of IS231A insertion specificity. Although both Tn4430 ends have identical sequences, one is strongly preferred to the other and the orientation of insertion into this end is not random. We demonstrate that this preference is determined by the flanking regions of the site. These regions display a conserved periodic organization of their sequence which, by conferring anisotropic flexibility, would induce the DNA to bend in a roughly 'S'-shaped structure centered on the target consensus. DNA conformation analysis by polyacrylamide gel electrophoresis indeed shows that the preferred target site of IS231A is flanked by DNA segments curved in opposite directions. We present a model in which DNA bendability and curvature would contribute to the positioning of IS231A transposase on the target DNA.