A Bacillus subtilis membrane-associated protein that binds specifically to the origin region of DNA replication may act as an inhibitor of DNA replication (J. Laffan and W. Firshein, Proc. Natl. Acad. Sci. USA 85:7452-7456, 1988). This protein, originally estimated to be 64 kDa, had a slightly lower molecular size (57 kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis during these studies. The size difference may be due to processing that results in modification of the protein. The protein can be extracted from both cytosol and membrane fractions, and the amounts in these fractions vary during the developmental cycle of B. subtilis. A complex pattern of expression in which significant levels were detected in spores was revealed; levels decreased dramatically during germination and increased after the first round of DNA replication. The decrease during germination was due to protease activity, as demonstrated by the addition of protease inhibitors and radioactive-labeling chase experiments. During vegetative growth, the protein levels increased until stationary phase, after which there was another decrease during sporulation. The decrease during sporulation may be partially due to sequestering of the protein into forespores, since as the putative repressor protein decreased in the mother cell, it increased in the forespores. However, protease activity was also involved in the decrease in the mother cell. The changes in expression of this protein are consistent with its role as a repressor of initiation of DNA replication. Additional studies, including sequence analysis and further antibody analysis, show that this protein is not a subunit of the pyruvate dehydrogenase complex. This relationship had been a possibility based upon the results of others (H. Hemila, A. Pavla, L. Paulin, S. Arvidson, and I. Palva, J. Bacteriol. 172:5052-5063, 1990).