The Na(+)-stimulated ATPase activity of Streptococcus faecalis was lost by washing the membranes with ethylenediaminetetraacetic acid (EDTA). ATPase activities of both the EDTA extract and the stripped membranes did not show any stimulation by Na+ ions. However, the Na(+)-stimulated ATPase was readily reconstituted by an incubation of these fractions combined. It was only reconstituted from the fractions prepared under the condition that the Na(+)-ATPase is amplified, and not from those boiled or digested by trypsin. Thus, the component of Na(+)-ATPase of this organism is capable of being released from the membranes.