Purified chicken skeletal muscle transverse tubule (T-tubule, TT) membrane preparations contain a very active Ca- or Mg-ATPase (EC 3.6.1.3) previously thought to be a T-system-specific marker enzyme. The function of the Mg-ATPase has not yet been determined although its prominent activity and concentration in junctional complexes supports a possible role in the excitation-contraction cycle. An essential component of the Mg-ATPase has been identified as a M(r) 85,000 glycoprotein (85k-GP). Polyclonal antibodies raised against the TT 85k-GP were specific and exhibited no cross-reactivity with other skeletal muscle proteins on immunoblots. Using this anti-85k-glycoprotein IgG, we have explored other chicken tissues to determine the tissue distribution of the 85k-GP. Antibody reactive polypeptides of M(r) 85,000 were found in gizzard smooth muscle, brain, heart, spleen, and lung tissue. The brain and smooth muscle membrane proteins were further purified and characterized for 85k-GP-associated Mg-ATPase activity. The brain and smooth muscle enzymes exhibited properties indistinguishable from the skeletal muscle TT-specific Mg-ATPase with regard to a series of activators and inhibitors, amino terminal amino acid sequences, and the effects of deglycosylation. The enzyme in all three tissues was inhibited by the diacylglycerol kinase inhibitor R 59022. Identification of the TT Mg-ATPase in gizzard smooth muscle has allowed the investigation of the Mg-ATPase membrane topology using isolated whole smooth muscle cells. The data support an ecto-orientation for the smooth muscle cell enzyme. Although the orientations of the brain and skeletal muscle enzymes have not been conclusively determined, the nearly identical properties of all three enzymes argues for an ecto-orientation of the active sites of these enzymes as well. The responsiveness of the three enzymes to regulatory lipids suggests that the ecto-Mg-ATPase may serve as a master switch controlling extracellular ATP concentrations and ligand accessibility to P1- and P2-purinoceptors. It is also proposed that the ecto-MgATPase may regulate ATP accessibility to ectoprotein kinases in a variety of tissues, and, in brain, the ecto-MgATPase may modulate the neurotransmitter role of ATP.