Previous studies which used intertypic reassortants of the wild-type reovirus serotype 1 Lang and the temperature-sensitive (ts) serotype 3 mutant clone tsA279 identified two ts lesions; one lesion, in the M2 gene segment, was associated with defective transmembrane transport of restrictively assembled virions (P. R. Hazelton and K. M. Coombs, Virology 207:46-58, 1995). In the present study we show that the second lesion, in the L2 gene segment, which encodes the 2 protein, is associated with the accumulation of a core-like particle defective for the 2 pentameric spike. Physicochemical, biochemical, and immunological studies showed that these structures were deficient for genomic double-stranded RNA, the core spike protein 2, and the minor core protein µ2. Core particles with the 2 spike structure accumulated after temperature shift-down from a restrictive to a permissive temperature in the presence of cycloheximide. These data suggest the spike-deficient, core-like particle is an assembly intermediate in reovirus morphogenesis. The existence of this naturally occurring primary core structure suggests that the core proteins 1, 3, and 2 interact to initiate the process of virion capsid assembly through a dodecahedral mechanism. The next step in the proposed capsid assembly model would be the association of the minor core protein µ2, either preceding or collateral to the condensation of the 2 pentameric spike at the apices of the primary core structure. The assembly pathway of the reovirus double capsid is further elaborated when these observations are combined with structures identified in other studies.