The 144-kDa lambda2 protein, a component of the transcriptionally active reovirus core particle, catalyzes the last three enzymatic activities for formation of the 5' cap 1 structure on the viral plus-strand transcripts. Limited evidence suggests it may also play a role in transcription per se. Particle-associated lambda2 forms pentameric turrets ("spikes") around the fivefold axes of the icosahedral core. To address the requirements for lambda2 in core functions other than the known functions in RNA capping, particles depleted of lambda2 were generated from cores in vitro by a series of treatments involving heat, protease, and ionic detergent. The resulting particles contained less than 5% of pretreatment levels of lambda2 but showed negligible loss of the other four core proteins or the 10 double-stranded RNA genome segments. Transmission cryo-electron microscopy (cryo-TEM) and scanning cryo-electron microscopy demonstrated loss of the lambda2 spikes from these otherwise intact particles. In functional analyses, the "spikeless cores" showed greatly reduced activities not only for RNA capping but also for transcription and nucleoside triphosphate hydrolysis, suggesting enzymatic or structural roles for lambda2 in all these activities. Comparison of the core and spikeless core structures obtained by cryo-TEM and three-dimensional image reconstruction revealed changes in the lambda1 core shell that accompany lambda2 loss, most notably the elimination of small pores that span the shell near the icosahedral fivefold axes. Changes in the shell may explain the reductions in transcriptase-related activities by spikeless cores.