We have studied the effects of the nonionic detergent C12E8 on Ca-ATPase enzymatic activity and oligomeric state (detected by time-resolved phosphorescence anisotropy, TPA) in skeletal and cardiac sarcoplasmic reticulum (SR). In skeletal, SR, C12E8 inhibits the CA-ATPase, both at high (micromolar and above) and low (submicromolar) Ca. In cardiac SR, C12E8 inhibits at high Ca but activates at low Ca. Thus C12E8 activates enzymatic activity only in cardiac SR and only under conditions (submicromolar Ca) where phospholamban (PLB) regulates (inhibits) the enzyme [Lu, Y.-Z., & Kirchberger, M.A. (1994) Biochemistry 33, 5056-5062]. TPA of skeletal SR at low and high Ca demonstrates that C12E8 induces aggregation of ATPase monomers and small oligomers. C12E8 also aggregates the Ca-ATPase in cardiac SR at high Ca. In cardiac SR at low Ca, the Ca-ATPase is already highly aggregated, and C12E8 partially dissociates these aggregates. Thus the TPA results provide a simple physical explanation for the functional effects: C12E8 inhibits the ATPase when it aggregates the enzyme (skeletal SR at high and low Ca; cardiac SR at high Ca), and the detergent activates when it dissociates ATPase oligomers (cardiac SR at low Ca). C12E8 stabilizes the E2P conformation of the Ca-ATPase with respect to the E2 conformation, and this stabilization is PLB-dependent. Both the physical and functional effects of C12E8 on the Ca-ATPase are PLB-dependent, with C12E8 reversing the effects of PLB. The results provide insight into the mechanism by which PLB regulates the Ca-ATPase in cardiac SR.