The transport of glucose by the solventogenic anaerobe Clostridium acetobutylicum was investigated. Glucose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity was detected in extracts prepared from cultures grown on glucose and extract fractionation revealed that both soluble and membrane components are required for activity. Glucose PTS activity was inhibited by the analogue methyl alpha-glucoside, indicating that the PTS enzyme II belongs to the glucose- glucoside (Glc) family of proteins. Consistent with this conclusion, labelled methyl alpha-glucoside was phosphorylated by PEP in cell- free extracts and this activity was inhibited by glucose. A single gene encoding a putative enzyme II of the glucose family, which we have designated glcG, was identified from the C. acetobutylicum ATCC 824 genome sequence. In common with certain other low-GC gram-positive bacteria, including Bacillus subtilis, the C. acetobutylicum glcG gene appears to be associated with a BglG-type regulator mechanism, as it is preceded by a transcription terminator that is partially overlapped by a typical ribonucleic antiterminator (RAT) sequence, and is downstream of an open reading frame that appears to encode a transcription antiterminator protein. This is the first report of a glucose transport mechanism in this industrially important organism.