The subcellular localization of membrane proteins in Bacillus subtilis was examined by using fluorescent protein fusions. ATP synthase and succinate dehydrogenase were found to localize within discrete domains on the membrane rather than being homogeneously distributed around the cell periphery as expected. Dual labelling of cells indicated partial colocalization of ATP synthase and succinate dehydrogenase. Further analysis using an ectopically expressed phage protein gave the same localization patterns as ATP synthase and succinate dehydrogenase, implying that membrane proteins are restricted to domains within the membrane. 3D reconstruction of images of the localization of ATP synthase showed that domains were not regular and there was no bias for localization to cell poles or any other positions. Further analysis revealed that this localization was highly dynamic, but random, implying that integral membrane proteins are free to diffuse two-dimensionally around the cytoplasmic membrane.