The busA (opuA) locus of Lactococcus lactis encodes a glycine betaine uptake system. Transcription of busA is osmotically inducible and its induction after an osmotic stress is reduced in the presence of glycine betaine. Using a genetic screen in CLG802, an Escherichia coli strain carrying a lacZ transcriptional fusion expressed under the control of the busA promoter, we isolated a genomic fragment from the L. lactis subsp. cremoris strain MG1363, which represses transcription from busAp. The cloned locus responsible for this repression was identified as a gene present upstream from the busA operon, encoding a putative DNA binding protein. This gene was named busR. Electrophoretic mobility shift and footprinting experiments showed that BusR is able to bind a site that overlaps the busA promoter. Overexpression of busR in L. lactis reduced expression of busA. Its disruption led to increased and essentially constitutive transcription of busA at low osmolarity. Therefore, BusR is a major actor of the osmotic regulation of busA in L. lactis.