pAM373 is a 36.7 kb conjugative plasmid in Enterococcus faecalis that encodes a response to a peptide sex pheromone, cAM373, secreted by plasmid-free (recipient) strains of enterococci. It was identified over 15 years ago as one of five plasmids in E. faecalis strain RC73 and was of interest because a related pheromone activity could be detected in culture supernatants of Staphylococcus aureus and Streptococcus gordonii. Because of increased clinical concern relating to the possibility of mobilizing vancomycin resistance determinants from enterococci, where they are becoming common, into pathogens such as S. aureus, efforts were initiated to characterize pAM373 further. The results of a complete nucleotide sequence determination of pAM373, as well as a genetic analysis of key genes related to regulation of the pheromone response, are reported here. With regard to determinants related to conjugation, the plasmid has a structural organization similar to other known pheromone-responsive plasmids such as pAD1, pCF10 and pPD1; however, there are several unique features. Although there are significant homologues relating to a pheromone-binding surface protein (TraC) and a negatively regulating protein (TraA), there is an absence of a determinant equivalent to traB of pAD1 (reduces endogenous pheromone) and a determinant for surface-exclusion protein. The precursor structure of the inhibitor peptide iAM373 was identified, and its determinant (iam373) was found to be about 500 nt upstream of an apparent transcription terminator t1. Tn917–lac insertion analyses provided interesting insights into aspects of control of the pheromone response and showed that, although the traA product is sensitive to pheromone, it appears to act differently from the traA homologue of pAD1.