ABSTRACT Rotaviruses are prototypical double-stranded RNA viruses whose triple-layered icosahedral capsid constitutes transcriptional machinery activated by the release of the external layer. To understand the molecular basis of this activation, we studied the structural interplay between the three capsid layers by electron cryo-microscopy and digital image processing. Two viral particles and four virus-like particles containing various combinations of inner (VP2)-, middle (VP6)-, and outer (VP7)-layer proteins were studied. We observed that the absence of the VP2 layer increases the particle diameter and changes the type of quasi-equivalent icosahedral symmetry, as described by the shift in triangulation number (T) of the VP6 layer (from T = 13 to T = 19 or more). By fitting X-ray models of VP6 into each reconstruction, we determined the quasi-atomic structures of the middle layers. These models showed that the VP6 lattices, i.e., curvature and trimer contacts, are characteristic of the particle composition. The different functional states of VP6 thus appear as being characterized by trimers having similar conformations but establishing different intertrimeric contacts. Remarkably, the external protein VP7 reorients the VP6 trimers located around the fivefold axes of the icosahedral capsid, thereby shrinking the channel through which mRNA exits the transcribing rotavirus particle. We conclude that the constraints arising from the different geometries imposed by the external and internal layers of the rotavirus capsid constitute a potential switch regulating the transcription activity of the viral particles.