Tn554, a transposon in Staphylococcus aureus that specifies resistance to erythromycin and spectinomycin, exhibits a high preference for a single chromosomal insertion site. If this site is already occupied by a copy of Tn554, the transposition of a second element is inhibited 100- to 1000-fold. This report defines the locus of the inhibitory activity and presents both a functional and a restriction map of Tn554. Fragments containing parts of Tn554 were cloned on an autonomously replicating plasmid. Those clones containing the "left" end of Tn554 strongly inhibited the transposition of an incoming, intact copy of Tn554. Analysis of deleted derivatives of these clones defined a locus tnpI, which is both necessary and sufficient for transpositional inhibition. This locus consists of the terminal 89 bp of the "left" end of Tn554. It is suggested that this terminal sequence acts to titrate a factor required for transposition.