Tn554 is a high-frequency, site-specific transposable element of Staphylococcus aureus which has integrative properties resembling those of temperate bacteriophages. Tn554 inserts at a unique chromosomal location, designated att554. att554 contains a core hexanucleotide sequence, 5'-GATGTA-3' (nucleotides numbered -3 to +3). Most of the time (greater than 99%) insertion occurs immediately 3' to this sequence; the resulting orientation of Tn554 to att554 is designated as the (+) orientation. Infrequent insertions immediately 5' to the core sequence result in the opposite, or (-) orientation. Mutational analysis of a cloned att554 site indicates that deletions extending from the left and ending at -15 or from the right ending between +8 and +12 reduced attachment site efficiency. Plasmids with deletions extending closer to the insertion site, although still retaining the core sequence from -3 to +3, were totally inactive. Tn554 insertions into partially active att554 sites retained normal site- and orientation-specificity with respect to att554, but they frequently contained abnormal sequences at the junction of att554 and the 3' end of Tn554. These data indicate that att554 contains a short nucleotide sequence essential for transposition and flanking sequences that greatly increase the frequency of recombination.