It is generally believed that sphingomyelin- and cholesterol-enriched microdomains can be isolated as detergent-resistant membranes (DRMs) from plasma membrane and organelle membranes. Here, we describe the isolation and characterization of microdomains from sarcoplasmic reticulum (SR) membranes. These SR-derived detergent-resistant membranes (SR-DRMs) enriched in sphingomyelin and cholesterol have a low buoyant density. Immunofluorescence microscopy of SR membranes shows the presence of caveolin-3 in the SR, known as a marker protein of caveolae at plasma membrane. We also demonstrated that significant amount of SERCAs together with caveolin-3 associates with SR-DRMs and are fully functional. Depletion of cholesterol caused the disruption of SR-DRMs. (c) 2006 Elsevier Inc. All rights reserved.