Preparations of sarcoplasmic reticulum ATPase made by conventional procedures, with over 85% of the protein consisting of one band in sodium dodecyl sulfate gel electrophoresis, were solubilized in Triton X-100 and separated on an Affi-Gel blue column. All the ATPase activity was eluted in a single fraction containing about 60% of the applied protein. This purified fraction required combination with about 1 mol of fluoresceinyl 5-isothiocyanate (FITC) for inactivation, whereas the original preparation was inactivated by reaction with about 0.6 mol of FITC/mol. The inactive protein retained on the column had an amino acid composition like that of the active protein. The separation on the Affi-Gel blue column provides a convenient procedure for preparation of more active ATPase. The rate of inactivation of the ATPase solubilized in detergent-containing solutions was measured at different protein concentrations. The t1/2 for inactivation was proportional to the square root of the protein concentration. Results are consistent with inactivation proceeding through a small fraction of monomeric ATPase present.