An early step in the utilization of starch by Bacteroides thetaiotaomicron is the binding of starch to the bacterial surface. Four starch-associated outer membrane proteins of B. thetaiotaomicron that have no starch-degrading activity have been identified. Two of these, SusC and SusD, have been shown by genetic analysis to be required for starch binding. In this study, we provide the first biochemical evidence that these two proteins interact physically with each other. Both formaldehyde cross-linking and nondenaturing gel electrophoresis experiments showed that SusC and SusD interact to form a complex. Two other proteins encoded by genes in the same operon, SusE and SusF, proved not to be essential for starch utilization and actually decreased starch binding when they were present along with SusC and SusD. Consistent with this, nondenaturing gel analysis revealed that in a strain producing SusC, SusD, and SusE, the SusCD complex was partially destabilized. The strain producing SusC, SusD, and SusE also grew more slowly on starch than a strain producing SusC, SusD, SusE, and SusF (mu(max), 0.29 and 0.37/h, respectively). Thus, SusE appears to interact with the SusCD complex. SusE also interacts with SusF, because SusE was less susceptible to proteinase K digestion when SusF was present, and nondenaturing gel analysis detected a complex formed by these two proteins. Our results indicate that SusC, SusD, SusE, and SusF form a protein complex in the outer membrane but that SusE and SusF are dispensable members of this complex.