The Streptococcus pneumoniae MalR protein regulates the transcription of two divergent operons, malXCD and malMP, involved in maltosaccharide uptake and utilization, respectively. MalR belongs to the LacI-GalR family of transcription repressors. The protein binds specifically to two operator sequences in the intergenic region between these operons. The affinity of MalR for the malMP binding sequence is higher than for the malXCD site. Results obtained in vivo using transcriptional fusions with reporter genes indicate low repression level of malXCD by MalR when compared with malMP. This behavior may be correlated with the existence of separate induction pathways for maltose, maltotriose, and maltotetraose. The similarities found at the operator sequences and binding domains for MalR and enterococcal repressor proteins suggest that the pneumococcal maltosaccharide regulation system is closely related to several Gram-negative metabolic pathways, but not to the structurally similar Escherichia coli maltose regulon.