The genomes of the rotaviruses consist of 11 segments of double-stranded RNA. During RNA replication, the viral plus-strand RNA serves as the template for minus-strand RNA synthesis. To characterize the kinetics of RNA replication, the synthesis and steady-state levels of viral plus- and minus-strand RNA and double-stranded RNA in simian rotavirus SA11-infected MA104 cells were analyzed by electrophoresis on 1.75% agarose gels containing 6 M urea (pH 3.0). Synthesis of viral plus-strand and minus-strand RNAs was detected initially at 3 h postinfection. The steady-state levels of plus- and minus-strand RNAs increased from this time until 9 to 12 h postinfection, at which time the levels were maximal. Pulse-labeling of infected cells with [3H]uridine showed that the ratio of plus- to minus-strand RNA synthesis changed during infection and that the maximal level of minus-strand RNA synthesis occurred several hours prior to the peak of plus-strand RNA synthesis. No direct correlation was found between the levels of plus-strand and minus-strand RNA synthesis in the infected cell. Pulse-labelling studies indicated that both newly synthesized and preexisting plus-strand RNA can act as templates for minus-strand RNA synthesis throughout infection. Studies also showed that less than 1 h was required between the synthesis of minus-strand RNA in vivo and its release from the cell within virions.