We provide a functional and regulatory analysis of glcP, encoding the major glucose transporter of Streptomyces coelicolor A3(2). GlcP, a member of the Major Facilitator Superfamily (MFS) of bacterial and eucaryotic sugar permeases, was found to be encoded twice at two distinct loci, glcP1 and glcP2, located in the central core and in the variable right arm of the chromosome respectively. Heterologous expression of GlcP in Escherichia coli led to the full restoration of glucose fermentation to mutants lacking glucose transport activity. Biochemical analysis revealed an affinity constant in the low-micromolar range and substrate specificity for glucose and 2-deoxyglucose. Deletion of glcP1 but not glcP2 led to a drastic reduction in growth on glucose reflected by the loss of glucose uptake. This correlated with transcriptional analyses, which showed that glcP1 transcription was strongly inducible by glucose, while glcP2 transcripts were barely detectable. In conclusion, GlcP, which is the first glucose permease from high G+C Gram-positive bacteria characterized at the molecular level, represents the major glucose uptake system in S. coelicolor A3(2) that is indispensable for the high growth rate on glucose. It is anticipated that the activity of GlcP is linked to other glucose-mediated phenomena such as carbon catabolite repression, morphogenesis and antibiotic production.