Mutants (glk) of Streptomyces coelicolor A3(2) that are resistant to the non-utilizable glucose analogue 2-deoxyglucose are deficient in glucose kinase activity, defective in glucose repression, and usually unable to utilize glucose. A 2.9 kb BclI fragment, previously shown to restore a wild-type phenotype to a glk deletion mutant that lacks the entire segment, contains two complete open reading frames that would encode proteins of 20.1 kDa (ORF2) and 33.1 kDa (ORF3). ORF3 is transcribed from its own promoter, and also from a promoter that initiates transcription upstream of ORF2. A derivative of the temperate phage phi C31 containing ORF3 alone restored a wild-type phenotype when used to lysogenize the deletion mutant. The product of ORF3 is homologous to members of a family of repressor proteins encoded by xylR in Bacillus subtilis and Lactobacillus pentosus, and by nagC in Escherichia coli. Although this might suggest that ORF3 encodes a positive activator for glucose kinase, rather than the enzyme itself, ORF3 restored the ability to metabolize glucose to an E. coli glk mutant, and activity gels of cell extracts of E. coli containing ORF3 cloned in the pT7-7 expression vector demonstrated that the ORF3 product has glucose kinase activity.