Growth, morphology, glutamine synthetase activity, cytochrome C oxidase activity and respiratory activity of rat brain cultures enriched in astrocytes were studied during four weeks in culture. Two different polarographic methods were used for measurement of respiratory activity: one newly developed perfusion method leaving the cellular monolayer morphologically intact and still attached to the culture dish, and one traditional stirring method involving the removal of cells from the culture vessel. Regardless of the method used, a stable respiratory activity was registrated throughout the four weeks of culturing. Also the cytochrome C oxidase activity remained unchanged. In perfusion all absolute values for respiration were found to be higher than those obtained with the stirring method. The use of the stirring technique resulted in a doubling of oxygen consumption upon succinate addition. No such effect was seen in perfusion. It can thus be concluded that the removal of cultured astrocytic cells from their substratum alters their respiratory activity and their response to added substrates.