Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub‐domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria‐associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing the substantial protein yields from HCMV‐infected primary fibroblasts and from transfected HeLa cells. Moreover, this unit includes a protocol that allows visualization of the mitochondria network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients. Curr. Protoc. Cell Biol. 37:3.27.1‐3.27.23. © 2007 by John Wiley & Sons, Inc.