The segmented double-stranded (ds)RNA genome of the rotaviruses is replicated asymmetrically with viral mRNA serving as the template for minus-strand RNA synthesis. To identify intermediate structures in rotavirus replication, subviral particles (SVPs) purified from the cytoplasm of simian rotavirus SA11-infected cells were assayed for RNA polymerase activity in a cell-free system that supports viral RNA replication. Intact SVPs containing newly made RNA were resolved by electrophoresis under nondenaturing conditions on 0.6% agarose gels (50 mM Tris-glycine, pH 8.8). This gel system was found to separate without disrupting SA11 single- and double-shelled virions and virion-derived core particles. SVPs from the cell-free system that contained newly made dsRNA migrated in the agarose gels at positions between virion-derived cores and intermediate of single- and double-shelled virions. SVPs containing newly made dsRNA were eluted from the gel and analyzed for protein content by electrophoresis on polyacrylamide gels. The results showed that three distinct types of replication intermediates (RIs) were present in SA11-infected cells. The smallest intermediate (precore RI, 45 nm, 220 S) contained the structural proteins VP1, VP3, and VP9 and the nonstructural proteins NS53, NS35, and NS34. A second intermediate (core RI, 60 nm, 310 S) contained the core proteins VP1, VP2, and VP3 and the proteins VP9, NS35 and NS34. The largest RI (single-shelled RI, 75 nm, 420 S) contained the inner shell proteins VP1, VP2, VP3, and VP6 and the proteins VP9, NS35 and NS34. Analysis of the formation and turnover of RIs in infected cells pulse-labeled with 35S-amino acids supports a hypothesis that rotavirus single-shelled particles are assembled in vivo by the sequential addition of VP2 and VP6 to precore RIs consisting of VP1, VP3, VP9, NS35, and NS34.