Transverse tubule membranes isolated from rabbit fast skeletal muscle contain a very active Mg2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3). This enzyme is very sensitive to inactivation by most detergents. However, after solubilization with either lysolecithin or digitonin, the Mg2+-ATPase can be purified in active form. Using a combination of selective solubilization followed by lectin affinity chromatography, ion-exchange chromatography, and native gel electrophoresis, the Mg2+-ATPase has been purified to near homogeneity. A prominent band with molecular mass of 105 kDa is observed when the purified protein is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified 105-kDa Mg2+-ATPase protein is not structurally similar to the sarcoplasmic reticulum Ca2+-ATPase protein, as evidenced by very different cyanogen bromide peptide maps and amino acid compositions. The structural dissimilarities are complemented by functional differences observed between the Ca2+- and Mg2+-ATPases, including differential susceptibility to proteases, chemical modification reagents, and inactivation by fluorescein isothiocyanate and vanadate. All these data taken together demonstrate that the Mg2+-ATPase is a unique protein with little, if any, structural similarity to the sarcoplasmic reticulum Ca2+-ATPase or to other related enzymes such as mammalian kidney (Na,K)-ATPase or gastric mucosal (H,K)-ATPase.