Three Streptococcus faecalis tetracycline-resistance conjugal plasmids carrying Tn916 were studied by using protoplast fusions, protoplast transformations, and membrane-filter matings between S. faecalis and Staphylococcus aureus. Two of these plasmids (pAD1: :Tn916 and pPD5: :Tn916)were capable of transfer to S. aureus by membrane-filter matings. These plasmids remained intact and expressed Tc('r) in S. aureus. Further transfers by membrane-filter matings among strains of S. aureus occurred only with pAD1: :Tn916, which transferred into the restrictionless lytic group II S. aureus strain 879R4. The third plasmid, pPD1: :Tn916, could not be demonstrated to transfer from S. faecalis to S. aureus by membrane-filter matings, protoplast fusions, or protoplast transformations;Transposition of Tn916 from pAD1: :Tn916 to the chromosome of lytic group I S. aureus has been demonstrated. DNA hybridization profiles obtained frm Southern transfers showed that Tn916 inserted at various sites on the S. aureus chromosome. In two instances (S. aureus ISP823 and S. faecalis ISP871), two copies of Tn916 were shown to be present at separate chromosomal sites. Mapping data obtained by transformation showed that Tn916 chromosomal insertions occurred near ilv-129, tyrB282, ala-126, fus-149, and thrB-trpE. Identical results were obtained when biotinylated pAD1: :Tn916 and 32P-labelled pAD1: :Tn916 probes were used to detect Tn916-containing sequences in hybridization experiments;pAD1: :Tn916 and pPD5: :Tn916, when transformed into protoplasts of S. aureus, gave rise to transformants containing the streptococcal plasmid alone, the streptococcal plasmid plus pPQ12, or pPQ12 alone. pPQ12 is a 2.8-Mdal Tc('r) plasmid in incompatibility group 3, and has the same restriction pattern and incompatibility grouping as pPQ3 and pSA0301, two representative naturally-occurring Tc('r) plasmids of S. aureus. pPQ12 did not hybridize to pAD1: :Tn916 but did hybridize to several naturally occurring Tc('r) plasmids of S. aureus as well as to lytic group I, II, and III S. aureus chromosomal DNA. It was concluded that pPQ12 is integrated in the S. aureus chromosome and does not express Tc('r) from the chromosomal site.