The high-GC Gram-positive actinomycete Corynebacterium glutamicum is commercially exploited as a producer of amino acids that are used as animal feed additives and flavor enhancers. Despite its beneficial role, carbon metabolism and its possible influence on amino acid metabolism is poorly understood. We have addressed this issue by analyzing the phosphotransferase system (PTS), which in many bacteria controls the flux of nutrients and therefore regulates carbon metabolism. The general PTS phosphotransferases enzyme I (EI) and HPr were characterized by demonstration of PEP-dependent phosphotransferase activity. An EI mutant exhibited a pleiotropic negative phenotype in carbon utilization. The role of the PTS as a major sugar uptake system was further demonstrated by the finding that glucose and fructose negative mutants were deficient in the respective enzyme II PTS permease activities. These carbon sources also caused repression of glutamate uptake, which suggests an involvement of the PTS in carbon regulation. The observation that no HPr kinase/phosphatase could be detected suggests that the mechanism of carbon regulation in C. glutamicum is different to the one found in low-GC Gram-positive bacteria.