Four temperature-sensitive mutations in the divIB gene of Bacillus subtilis have been localized to the region corresponding to the C-terminal half of the 263-residue DivIB protein. Antiserum was raised to the 80% C-terminal portion lying on one side of a putative transmembrane (hydrophobic) segment, and used to examine aspects of the nature and localization of the DivIB protein in the cell. A single DivIB species of a size equal to the full-length protein encoded by the divIB gene was detected in wild-type cells. Cell fractionation studies established that DivIB is associated preferentially with the cell envelope (membrane plus cell wall), with approximately 50% being released into solution upon treatment of cells with lysozyme under conditions that yield protoplasts. Of the remaining 50%, approximately half remained firmly associated with the membrane fraction. On the basis of the 'positive-inside rule' of von Heijne (1986) it is suggested that the topology of membrane-bound DivIB is such that the long C-terminal portion is directed to the outside and the smaller N-terminal portion to the inside of the cell. DivIB in protoplasts was rapidly degraded by proteinase K under conditions where there was no general proteolysis of the cytoplasmic proteins. This is consistent with its absence from the cytoplasm, and with the predicted membrane topology. Septum positioning in a divIB null mutant, which grows as filaments at temperatures of 30 degrees C and below, was found to be normal. It appears that DivIB is needed for achieving the appropriate rate of initiation of septum formation at normal division sites. It is proposed that the C-terminal portion of DivIB, localized on the exterior surface of the membrane and in juxtaposition to the peptidoglycan, normally interacts with another protein (or proteins) to initiate septum formation.