ABSTRACT Corynebacterium glutamicum can grow on l -lactate as a sole carbon and energy source. The NCgl2816- lldD operon encoding a putative transporter (NCgl2816) and a quinone-dependent l -lactate dehydrogenase (LldD) is required for l -lactate utilization. DNA affinity chromatography revealed that the FadR-type regulator LldR (encoded by NCgl2814) binds to the upstream region of NCgl2816- lldD . Overexpression of lldR resulted in strongly reduced NCgl2816- lldD mRNA levels and strongly reduced LldD activity, and as a consequence, a severe growth defect was observed in cells grown on l -lactate as the sole carbon and energy source, but not in cells grown on glucose, ribose, or acetate. Deletion of lldR had no effect on growth on these carbon sources but resulted in high NCgl2816- lldD mRNA levels and high LldD activity in the presence and absence of l -lactate. Purified His-tagged LldR bound to a 54-bp fragment of the NCgl2816- lldD promoter, which overlaps with the transcriptional start site determined by random amplification of cDNA ends-PCR and contains a putative operator motif typical of FadR-type regulators, which is ⁻¹ TNGTNNNACNA ¹⁰ . Mutational analysis revealed that this motif with hyphenated dyad symmetry is essential for binding of LldD to the NCgl2816- lldD promoter. l -Lactate, but not d -lactate, interfered with binding of LldR ^His to the NCgl2816- lldD promoter. Thus, during growth on media lacking l -lactate, LldR represses expression of NCgl2816- lldD . In the presence of l -lactate in the growth medium or under conditions leading to intracellular l -lactate accumulation, the l -lactate utilization operon is induced.