A Na+ and K+ stimulated, Mg2+-requiring ATPase has been demonstrated in the homogenate and microsomal fractions of aortic smooth muscle. Tris–HCl buffer interfered with the Na+ plus K+ stimulatory effect revealing only a less specific nucleotide phosphatase activity. Also, in order to demonstrate stimulation with Na+ and K+, a soluble activating cofactor isolated from the 100 000 × g supernatant fraction was required. The highest activity was obtained from a density gradient prepared microsomal fraction with soluble activator added in the presence of 50 mM Na+, 2 mM K+, and 50 mM histidine buffer at pH 7.6, with 4 mM Mg-ATP as substrate. The greater part of the monovalent cation stimulated activity was due to a ouabain-insensitive Na+-stimulated ATPase activity. However, the remainder of the total stimulation was ouabain-sensitive and attributed to a Na+–K+-ATPase.