The Mu transpositional DNA recombination machinery selects target sites by assembling a protein-DNA complex that interacts with the target DNA and reacts whenever it locates a favorable sequence composition. Splicing of a transposon into the target generates a 5-bp duplication that reflects the original target site. Preferential usage of different target pentamers was examined with a minimal Mu in vitro system and quantitatively compiled consensus sequences for the most preferred and the least preferred sites were generated. When analyzed as base steps, preferences toward certain steps along the 5-bp target site were detected. We further show that insertion sites can be predicted on the basis of additively calculated base step values. Also surrounding sequences influence the preference of a given pentamer; a symmetrical structural component was revealed, suggesting potential hinges at and around the target site.