Many virulent strains of Enterococcus faecalis produce a two-subunit toxin, termed cytolysin. Cytolysin expression is regulated by one of the subunits (CylL_S″) through a quorum-sensing autoinduction mechanism. We found that when target cells are absent, the other subunit (CylL_L″) forms a complex with CylL_S″, blocking it from autoinducing the operon. When target cells are present, however, CylL_L″ binds preferentially to the target, allowing free CylL_S″ to accumulate above the induction threshold. Thus, enterococci use CylL_L″ to actively probe the environment for target cells, and when target cells are detected, allows the organism to express high levels of cytolysin in response.