After crossing the cellular membrane barrier during cell entry, most animal viruses must undergo further disassembly before initiating viral gene expression. In many cases, these disassembly mechanisms remain poorly defined. For this report, we examined a final step in disassembly of the mammalian reovirus outer capsid: cytoplasmic release of the central, delta fragment of membrane penetration protein mu1 to yield the transcriptionally active viral core particle. An in vitro assay with reticulocyte lysate recapitulated the release of intact delta molecules. Requirements for activity in this system were shown to include a protein factor, ATP, and Mg(2+) and K(+) ions, consistent with involvement of a molecular chaperone such as Hsc70. Immunodepletion of Hsc70 and Hsp70 impaired delta release, which was then rescued by addition of purified Hsc70. Hsc70 was associated with released delta molecules not only in the lysate but also during cell entry. We conclude that Hsc70 plays a defined role in reovirus outer capsid disassembly, during or soon after membrane penetration, to prepare the entering particle for gene expression and replication.