A novel teicoplanin-associated operon termed tcaR-tcaA-tcaB was identified by Tn917-mediated insertional mutagenesis. Resistance to teicoplanin rose 4-fold by insertional inactivation of tcaA or by deletion of the entire operon. tcaA encodes a hypothetical transmembrane protein with a metal-binding motif, possibly a sensor-transducer. tcaB codes for a membrane-associated protein, which has sequence homologies to a bicyclomycin resistance protein. The two genes are preceded by tcaR encoding a putative regulator with sequence homologies to the transcriptional regulator MarR. The fact that tcaA inactivation as well as deletion of tcaRAB produced the same increase in teicoplanin resistance confirmed the association of tcaRAB with teicoplanin susceptibility. Cotransductional crosses showed that the level of teicoplanin resistance produced by these insertions was strain-dependent and that in the methicillin-resistant strain COL, it was paired with a remarkable decrease in methicillin resistance. This allowed to postulate that tcaRAB may be involved in some way in cell wall biosynthesis, and that teicoplanin may interact with TcaA and/or TcaB either directly or indirectly.