The localization of ATP-hydrolysing activity in vegetative cells, spores and isolated membranes of Bacillus subtilis 168 was studied by a cytochemical method combined with electron microscopy. The activity was located mainly in the cytoplasmic membrane and the mesosomes, and was also found in the inner layer of the cell wall facing the cytoplasmic membrane. Activity was also detected in the cross-membranes of dividing cells and in spore coats. The product of the reaction was observed either as fine electron-dense granules incorporated into the membranes, or as high-contrast lead precipitates on the surfaces of the membranes.