All plus-strand RNA viruses replicate in association with cytoplasmic membranes of infected cells. The RNA replication complex of many virus families is associated with the endoplasmic reticulum membranes, for example, picorna-, flavi-, arteri-, and bromoviruses. However, endosomes and lysosomes (togaviruses), peroxisomes and chloroplasts (tombusviruses), and mitochondria (nodaviruses) are also used as sites for RNA replication. Studies of individual nonstructural proteins, the virus-specific components of the RNA replicase, have revealed that the replication complexes are associated with the membranes and targeted to the respective organelle by the ns proteins rather than RNA. Many ns proteins have hydrophobic sequences and may transverse the membrane like polytopic integral membrane proteins, whereas others interact with membranes monotopically. Hepatitis C virus ns proteins offer examples of polytopic transmembrane proteins (NS2, NS4B), a “tip-anchored” protein attached to the membrane by an amphipathic α-helix (NS5A) and a “tail-anchored” posttranslationally inserted protein (NS5B). Semliki Forest virus nsP1 is attached to the plasma membrane by a specific binding peptide in the middle of the protein, which forms an amphipathic α-helix. Interaction of nsP1 with membrane lipids is essential for its capping enzyme activities. The other soluble replicase proteins are directed to the endo-lysosomal membranes only as part of the initial polyprotein. Poliovirus ns proteins utilize endoplasmic reticulum membranes from which vesicles are released in COPII coats. However, these vesicles are not directed to the normal secretory pathway, but accumulate in the cytoplasm. In many cases the replicase proteins induce membrane invaginations or vesicles, which function as protective environments for RNA replication.