We identified α2, α1, and β1 isoforms of Na+/K+-ATPase in caveolae vesicles of bovine pulmonary smooth muscle plasma membrane. The biochemical and biophysical characteristics of the α2 β1 isozyme of Na+/K+-ATPase from caveolae vesicles were studied during solubilization and purification using the detergents 1,2-heptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C12E8), and Triton X-100, and reconstitution with the phospholipid dioleoyl-phosphatidylcholine (DOPC). DHPC was determined to be superior to C12E8, whereas C12E8 was better than Triton X-100 in the active enzyme yields and specific activity. Fluorescence studies with DHPC-purified α2 β1 isozyme of Na+/K+-ATPase elicited higher E1Na-E2 K transition compared with that of the C12E8- and Triton X-100-purified enzyme. The rate of Na+ efflux in DHPC-DOPC-reconstituted isozyme was higher compared to the C12E8-DOPC- and Triton X100-DOPC-reconstituted enzyme. Circular dichroism analysis suggests that the DHPC-purified α2β1 isozyme of Na+/K+-ATPase possessed more organized secondary structure compared to the C12E8- and Triton X-100-purified isozyme. © Springer Science+Business Media, LLC. 2008.