HSP72 is dramatically induced in the ovaries of vaccinia virus (VV)-infected mice and associates with VV proteins. In order to Investigate the role of HSP72 during vaccinia virus replication, we have constructed a recombinant vaccinia virus encoding the major inducible cellular HSP72 (VV-HSP72+) and examined the replication characteristics of this virus. VV-HSP72+ exhibited growth kinetics identical to and peak titers very similar to those of control viruses, both in vitro and in vivo. In particular, replication of VV-HSP72+ was identical to that of control viruses in the HSP72-negative cell line Y3.Ag.1.2.3, and overexpression of HSP72 had no effect on the virulence of VV infection in normal or immunocompromised mice. We conclude that while W infection results in the induction of the major inducible 72-kDa HSP, VV replication proceeds normally in the absence of this protein. It is unclear whether another celluar chaperone is required to facilitate virus replication in place of HSP72 in Y3.Ag.1.2.3 cells or whether HSP expression plays no role in virus replication, but is simply a component of the generalized stress response to virus infection.