The proper placement of the cell division site in Escherichia coli requires the site-specific inactivation of potential division sites at the cell poles in a process that is mediated by the MinC, MinD and MinE proteins. During the normal division cycle MinD plays two roles. It activates the MinC-dependent mechanism that is responsible for the inactivation of potential division sites and it also renders the division inhibition system sensitive to the topological specificity factor MinE. MinE suppresses the division block at the normal division site at mid-cell but not all cell poles, thereby ensuring the normal division pattern. In this study the MinD protein was purified to homogeneity and shown to bind ATP and to have ATPase activity. When the putative ATP binding domain of MinD was altered by site-directed mutagenesis, the mutant protein was no longer able to activate the MinC-dependent division inhibition system. Immunoelectron microscopy showed that MinD was located in the inner membrane region of the cell envelope. These results show that MinD is a membrane ATPase and suggest that the ATPase activity plays an essential role in the functions of the MinD protein during the normal division process.