Proteins from sarcoplasmic reticulum vesicles solubilized by a nonionic detergent were fractionated by use of a reactive red-120 agarose column. The Ca-ATPase was obtained in pure form by eluting the column with 400 microM adenyl 5'-yl imidodiphosphate, yielding an enzyme of almost twice the starting specific activity in a fraction containing half the initial protein. The conclusion that the ATPase comprises 50% of the sarcoplasmic reticulum vesicle protein agrees with estimates gained from densitometry using 7 1/2% Laemmli slab gels but not from densitometry using 7% Weber and Osborn slab gels. The mechanism of purification was found to be affinity chromatography, with the ATPase binding the reactive red-120 ligand in its nucleotide-binding site. The steady-state concentration of phosphorylated intermediate relative to the specific activity was found to be lower in the purified enzyme as compared to the starting vesicular enzyme.