The region upstream of the Escherichia coli bgl operon is an insertion hot spot for several transposons. Elements as distantly related as Tn1, Tn5, and phage Mu home in on this location. To see what characteristics result in a high-affinity site for transposition, we compared in vivo and in vitro Mu transposition patterns near the bgl promoter. In vivo, Mu insertions were focused in two narrow zones of DNA near bgl, and both zones exhibited a striking orientation bias. Five hot spots upstream of the bgl cyclic AMP binding protein (CAP) binding site had Mu insertions exclusively with the phage oriented left to right relative to the direction of bgl transcription. One hot spot within the CAP binding domain had the opposite (right-to-left) orientation of phage insertion. The DNA segment lying between these two Mu hot-spot clusters is extremely A/T rich (80%) and is an efficient target for insertion sequences during stationary phase. IS1 insertions that activate the bgl operon resulted in a decrease in Mu insertions near the CAP binding site. Mu transposition in vitro differed significantly from the in vivo transposition pattern, having a new hot-spot cluster at the border of the A/T-rich segment. Transposon hot-spot behavior and orientation bias may relate to an asymmetry of transposon DNA-protein complexes and to interactions with proteins that produce transcriptionally silenced chromatin.