Formation of virus-specific replicative complexes (RCs) in infected cells is one of the most intriguing and important processes that determine virus replication and ultimately their pathogenesis on the molecular and cellular levels. Alphavirus replication was known to lead to formation of so-called type 1 cytopathic vacuoles (CPV1s), whose distinguishing feature is the presence of numerous membrane invaginations (spherules) and accumulation of viral nonstructural proteins (nsPs) at the cytoplasmic necks of these spherules. These CPV1s, modified endosomes and lysosomes, were proposed as the sites of viral RNA synthesis. However, our recent studies have demonstrated that Sindbis virus (SINV)-specific, double-stranded RNA (dsRNA)- and nonstructural protein (nsP)-containing RCs are initially formed at the plasma membrane. In this new study, we present extensive evidence that (i) in cells of vertebrate origin, at early times postinfection, viral nsPs colocalize with spherules at the plasma membrane; (ii) viral dsRNA intermediates are packed into membrane spherules and are located in their cavities on the external surface of the plasma membrane; (iii) formation of the membrane spherules is induced by the partially processed nonstructural polyprotein P123 and nsP4, but synthesis of dsRNA is an essential prerequisite of their formation; (iv) plasma membrane-associated dsRNA and protein structures are the active sites of single-stranded RNA (ssRNA) synthesis; (v) at late times postinfection, only a small fraction of SINV nsP-containing complexes are relocalized into the cytoplasm on the endosome membrane. (vi) pharmacological drugs inhibiting different endocytotic pathways have either only minor or no negative effects on SINV RNA replication; and (vii) in mosquito cells, at any times postinfection, dsRNA/nsP complexes and spherules are associated with both endosomal/lysosomal and plasma membranes, suggesting that mechanisms of RC formation may differ in cells of insect and vertebrate origins. Copyright © 2010, American Society for Microbiology. All Rights Reserved.