The present study provides data on the properties of Ca2+-dependent Atpase of sarcoplasmic reticulum in states intermediary between the fully detergent-solubilized and vesicular form. After solubilization of ATPase vesicles by dodecyloctaoxyethylene glycol monoether (C12E8), the protein is mainly present as a monomer exhibiting enzymatic activity. Gel chromatography in presence or absence of Tween 80 gives rise to formation of oligomers of various size and smaller amounts of monomeric ATPase. Only the oligomeric species retain enzymatic activity (half-life, 3 to 4 days), while the gel chromatographic monomer is enzymatically inactive. Teteramers or trimers of ATPase, containing approximately 22 mol of phospholipid/mol of ATPase, are the smallest enzymatically active units after gel chromatography. Formation of larger sized particles and vesicles of ATPase appears to depend on the presence of sufficient lipid to make a cohesion between the tetrameric or trimeric units. The protein appears to be partially deaggregated by a relatively high Tween 80 concentration in the eluant (0.5 mg/ml) and under these conditions, phospholipid binding is reduced to a low level (approximately 11 mol/mol of protein). The data indicate that any bonds between ATPase polypeptide chains are easily disrupted by detergent and that lipid also may play a role in mediating contact between individual polypeptide chains in the tetrameric or trimeric units. Phospholipid analysis and exchange experiments indicate that the phospholipid left on ATPase after solubilization has a similar composition to that of the whole membrane. The binding of Tween 80 by soluble ATPase above the critical micellar concentration is 0.23 to 0.29 g/g of protein. The inactive monomer of ATPase binds phospholipid and Tween 80 to about the same extent, but has a slightly different circular dichroism spectrum, than oligomeric ATPase.