The hemB gene of Escherichia coli has been identified as a hot spot for the insertion of the transposable element IS2. The insertional specificity of IS2 is still unclear. This study reports on the attempt to sequence a statistically significant number of insertions in hemB, in order to determine whether there might be a basis for future studies to determine a molecular basis of IS2 insertional specificity. The results indicate that IS2 inserts in a non-random manner into a 240bp segment at the 5' end of the gene (region I). Twenty-one of 24 insertions occurred in region I. Three insertions have been identified in the two middle 250bp segments of the 975bp gene, and none in the 3' terminal segment. A seventeen bp sequence showing 88.2% identity with a segment of IS2, 221bp from the 3' terminus has been identified in region I. Four instances of repeated insertion between the same pair of nucleotides have been observed at four different sites.