Hexanol at 7 °C stimulates the activity of the Ca-ATPase of sarcoplasmic reticulum (SR). Time-resolved phosphorescence spectroscopy studies of SR whose Ca-ATPase is covalently labeled with erythrosin isothiocyanate (ERITC) indicate that at 7 °C hexanol (1) causes a concentration-dependent increase in the rate of decay of phosphorescence anisotropy, (2) causes larger oligomers of Ca-ATPase to dissociate into smaller oligomers, and (3) increases the rotational mobility of Ca-ATPase in all its oligomeric states. Electron paramagnetic resonance (EPR) spectroscopy of spin-labeled stearic acid (SASL) in SR suggests that at 7 °C hexanol diminishes the fraction of SR lipids in the boundary lipid domain and disorders and fluidizes both the boundary lipid and the unrestricted lipid domain. In protein-free liposomes of extracted SR lipids hexanol increases fluidity and decreases order to a greater extent near the center of the lipid bilayer than near the polar head groups. At 25 °C hexanol has biphasic effects on Ca-ATPase activity: at 10 and 20 mM hexanol increases activity, but at 30 mM and especially at 40 mM there is inhibition of Ca-ATPase activity. The influence of hexanol at 25 °C on the oligomeric state of Ca- ATPase is also biphasic. At 10 and 20 mM, hexanol promotes the dissociation of larger oligomers into smaller ones, whereas at higher concentrations, 30 and 40 mM, hexanol causes larger oligomers to be formed from smaller ones. Lidocaine at 25 °C inhibits Ca-ATPase activity and causes dramatic slowing of the decay of phosphorescence anisotropy of ERITC-labeled SR by causing the formation of larger oligomers of Ca-ATPase from smaller ones. In protein-free liposomes of SR lipids at 25 °C, lidocaine disorders and fluidizes the acyl chains near the center of the bilayer (as did hexanol), but has opposite effects near the polar head groups. The opposite effects of hexanol and lidocaine on the oligomeric state of the SR Ca-ATPase provide a new molecular explanation for the opposite effects of hexanol and lidocaine on the activity of the Ca-ATPase. We conclude that the biphasic effects of hexanol on the activity of Ca-ATPase can be accounted for by biphasic effects of hexanol on the oligomeric state of the Ca-ATPase. This study supports the view that anesthetics can alter interactions between membrane proteins.