The complete nucleotide sequence of the Staphylococcus aureus transposon Tn554, which encodes resistance to erythromycin and spectinomycin, was determined by the dideoxy chain termination method. The transposon was found to be 6691 bp in length and to contain six open reading frames of greater than 125 amino acids. Small insertion and deletion mutations were obtained in each of these by in vitro mutagenesis at restriction endonuclease cleavage sites and the mutants characterized with respect to transposition functions and antibiotic resistance markers. Three of the reading frames, designated tnpA, tnpB and tnpC, encode functions that are required for transposition of Tn554; genetic analysis indicated that these three genes define distinct complementation groups of transposition-defective mutants. Two of the open reading frames correspond to the resistance determinants spc and ermA, the sixth, designated ORF, has no known function. Tn554-specific peptides corresponding to tnpA, and spc were identified in a coupled transcription-translation system in vitro.