Bacteroides thetaiotaomicron uses starch as a source of carbon and energy. Early steps in the pathway of starch utilization, such as starch binding and starch hydrolysis, are encoded by sus genes, which have been characterized previously. The sus structural genes are expressed only if cells are grown in medium containing maltose or higher oligomers of glucose. Regulation of the sus structural genes is mediated by SusR, an activator that is encoded by a gene located next to the sus structural genes. A strain with a disruption in susR cannot grow on starch but can still grow on maltose and maltotriose. A search for transposon-generated mutants that could not grow on maltose and maltotriose unexpectedly located a gene, designated malR, which regulates expression of an alpha-glucosidase not controlled by SusR. Although a disruption in susR did not affect expression of the malR controlled gene, a disruption in malR reduced expression of the sus structural genes. Thus, MalR appears to participate with SusR in regulation of the sus genes. Results of transcriptional fusion assays and reverse transcription-PCR experiments showed that malR is expressed constitutively. Moreover, multiple copies of malR provided on a plasmid (5 to 10 copies per cell) more than doubled the amount of alpha-glucosidase activity in cell extracts. Our results demonstrate that the starch utilization system of B. thetaiotaomicron is controlled on at least two levels by the regulatory proteins SusR and MalR.