The putative two-component system BfrAB is involved in Streptococcus gordonii biofilm development. Here, we provide evidence that BfrAB regulates the expression of bfrCD and bfrEFG, which encode two ATP-binding cassette (ABC) transporters, and bfrH, which encodes a CAAX amino-terminal protease family protein. BfrC and BfrE are ATP-binding proteins, and BfrD, BfrF and BfrG are homologous membrane-spanning polypeptides. Similarly, BfrABss, the BfrAB homologous system in Streptococcus sanguinis, controls the expression of two bfrCD-homologous operons (bfrCDss and bfrXYss), a bfrH-homologous gene (bfrH1ss) and another CAAX amino-terminal protease family protein gene (bfrH2ss). Furthermore, we demonstrate that the purified BfrA DNA-binding domain from S. gordonii binds to the promoter regions of bfrCD, bfrEFG, bfrH, bfrCDss, bfrXYss and bfrH1ss in vitro. Finally, we show that the BfrA DNA-binding domain recognizes a conserved DNA motif with a consensus sequence of TTTCTTTAGAAATATTTTAGAATT. These data suggest, therefore, that S. gordonii BfrAB controls biofilm formation by regulating multiple ABC-transporter systems.