The ability of Streptococcus mutans to catabolize cellobiose, a beta-linked glucoside generated during the hydrolysis of cellulose, is shown to be regulated by a transcriptional regulator, CelR, which is encoded by an operon with a phospho-beta-glucosidase (CelA) and a cellobiose-specific sugar phosphotransferase system (PTS) permease (EII(Cel)). The roles of CelR, EII(Cel) components, and certain fructose/mannose-PTS permeases in the transcriptional regulation of the cel locus were analyzed. The results revealed that (i) the celA and celB (EIIB(Cel)) gene promoters require CelR for transcriptional activation in response to cellobiose, but read-through from the celA promoter contributes to expression of the EII(Cel) genes; (ii) the EII(Cel) subunits were required for growth on cellobiose and for transcriptional activation of the cel genes; (iii) CcpA plays little direct role in catabolite repression of the cel regulon, but loss of specific PTS permeases alleviated repression of cel genes in the presence of preferred carbohydrates; and (iv) glucose could induce transcription of the cel regulon when transported by EII(Cel). CelR derivatives containing amino acid substitutions for five conserved histidine residues in two PTS regulatory domains and an EIIA-like domain also provided important insights regarding the function of this regulator. Based on these data, a model for the involvement of PTS permeases and the general PTS proteins enzyme I and HPr was developed that reveals a critical role for the PTS in CcpA-independent catabolite repression and induction of cel gene expression in S. mutans.