We recently developed an in vitro transcapsidation system in which infectivity of single-shelled (ss) rotavirus particles was successfully rescued (Chen and Ramig, Virology ). Here, we report the rescue of infectivity of rotavirus core particles using virus strain B223 (G serotype 10) as the core donor and strain SA11-4F (G3) as the capsid donor. Core particles of B223 were obtained by CaCl2 treatment of B223 ss-particles followed by isopycnic CsCl gradient centrifugation. Inner capsid protein VP6 of SA11-4F was prepared by CaCl2 treatment of SA11-4F ss-particles, followed by removal of core particles by two rounds of centrifugation. Outer capsid proteins VP4 and VP7 of SA11-4F were prepared by EDTA treatment of ds-particles, followed by three rounds of centrifugation to remove ss-particles and minimize residual infectivity. No infectivity (< 3 PFU/ml) was detectable in any of the donor preparations. Transcapsidated ss-particles were obtained by mixing B223 core particles and a 5-fold excess of SA11-4F VP6 at neutral pH. The formation of transcapsidated ss-particles was confirmed by electron microscopy, protein composition analysis, and density determination. Along with the formation of ss-particles by in vitro transcapsidation, some infectivity was also detected and transcriptase activity was reconstituted. Semi-purified transcapsidated ss-particles were then mixed with SA11-4F outer capsid proteins VP4 and VP7 at acidic pH to obtain transcapsidated ds-particles, as described previously. The formation of ds-like particles was also confirmed by electron microscopy, protein composition, and density determination. As the result of formation of transcapsidated ds-like particles, viral infectivity increased significantly (80-fold) relative to that of transcapsidated ss-particles. The infectivity of transcapsidated ds-particles was neutralized by polyclonal anti-SA11 serum, but not by polyclonal anti-B223 serum. The transcapsidated particles formed small plaques like B223 (core donor), and all the progeny plaques contained B223 genomes. These results demonstrate that the infectivity of rotavirus core particles can be rescued by sequential addition of inner and outer capsid proteins in vitro.