NdgR, a global regulator in soil-dwelling and antibiotic-producingStreptomyces, is known to regulate branched-chain amino acid metabolism by binding to the upstream region of synthetic genes. However, its numerous and complex roles are not yet fully understood. To more fully reveal the function of NdgR, phospholipid fatty acid (PLFA) analysis with gas chromatography-mass spectrometry (GC-MS) was used to assess the effects of anndgRdeletion mutant ofStreptomyces coelicolor. The deletion ofndgRwas found to decrease the levels of isoleucine- and leucine-related fatty acids but increase those of valine-related fatty acids. Furthermore, the defects in leucine and isoleucine metabolism caused by the deletion impaired the growth ofStreptomycesat low temperatures. Supplementation of leucine and isoleucine, however, could complement this defect under cold shock condition. NdgR was thus shown to be involved in the control of branched-chain amino acids and consequently affected the membrane fatty acid composition inStreptomyces. While isoleucine and valine could be synthesized by the same enzymes (IlvB/N, IlvC, IlvD, and IlvE),ndgRdeletion did not affect them in the same way. This suggests that NdgR is involved in the upper isoleucine and valine pathways, or that its control over them differs in some respect.