Fragments, generated by restriction enzyme digestion, of the 58-kilobase Enterococcus (Streptococcus) faecalis tetracycline resistance plasmid pCF10 were cloned and introduced into Escherichia coli and E. faecalis to characterize the pheromone-inducible conjugation system encoded by this plasmid. Western blot (immunoblot) analyses revealed that a 130-kilodalton (kDa) antigen, identical to the Tra130 antigen shown previously to be involved in pCF10-mediated pheromone-inducible surface exclusion, was produced by both bacterial hosts carrying the recombinant plasmid pINY1825 (cloned EcoRI C fragment). Both bacterial hosts carrying pINY1825 also produced various amounts of immunologically related 118- to 125-kDa antigens (designated pre-Tra130) that resembled antigens produced by E. faecalis cells carrying pCF10. An additional 150-kDa antigen, Tra150, probably involved in pheromone-induced cellular aggregation, was produced by Escherichia coli and E. faecalis hosts carrying pINY1801 (cloned EcoRI C and E fragments). The coding sequences for the Tra150 and Tra130 antigens were further localized in the TRA region of pCF10 by transposon insertion mutagenesis. Western blot analyses of the recombinant strains, and of strains carrying derivatives of pCF10 or various recombinant plasmids containing Tn5 or Tn917 insertions, suggested that the portion of pCF10 comprising the tra3 through -6 segments (previously defined by Tn917 insertional mutagenesis) contained several genes that are involved in regulating the synthesis of Tra130 and Tra150.