The Bacillus subtilis proteins DnaD and DnaB are essential for replication initiation and are conserved in low G+C content Gram-positive bacteria. Previous work indicated that DnaD and DnaB are involved in helicase loading during the process of restarting stalled replication forks. We have investigated the roles of DnaD and DnaB in replication initiation at oriC in vivo. Using chromatin immunoprecipitation (ChIP), we found that DnaD and DnaB functions are needed to load the replicative helicase at oriC. To investigate further the functions of DnaD and DnaB in replication initiation, we isolated and characterized suppressors of the temperature sensitivity of dnaD and dnaB mutant cells. In both cases, we isolated the identical missense mutation in dnaB, dnaBS371P. Using yeast two-hybrid analysis, we found that dnaBS371P uncovers a previously undetected physical interaction between DnaD and DnaB. We also found that DnaBS371P constitutively recruits DnaD to the membrane fraction of cells, where DnaB and oriC are enriched. Phenotypes of cells expressing DnaBS371P are consistent with aberrant replication control. We hypothesize that B. subtilis regulates replication initiation by regulating a physical interaction between two proteins essential for helicase loading at chromosomal origins.