Following infection, many secreted poxvirus proteins are able to modulate the host immune response through interactions with cytokines or components of the complement pathway. A comparison of the secreted protein profiles from cells infected with vaccinia Western Reserve (VV-WR), cowpox virus Brighton strain, or rabbitpox virus (RPV) showed an abundant 35-kDa protein present only in the supernatants from RPV-infected cells. The gene encoding this protein was identified and mapped by N-terminal sequencing of the protein. Examination of the predicted amino acid sequence showed it to be identical to the 35-kDa secreted protein of the Lister strain of vaccinia virus described by Patel et al. (1990, J. Gen. Virol. 71, 2013-2021). The counterpart of this gene in the commonly studied VV-WR strain is truncated and encodes a 7.5-kDa protein under control of the well-characterized p7.5 promoter. While nonessential for replication in cell culture, conservation of this gene in at least two orthopoxvirus strains suggested that this protein might play an important role in vivo. Following intranasal inoculation of Balb/c mice at several doses (10(3), 10(4), or 10(5) PFU), a mutant of RPV lacking a functional 35-kDa gene (RPV delta 35) appeared to induce an earlier onset and more severe illness at low, sublethal doses (10(3) PFU) than was observed with wild-type (wt) RPV. At higher doses (10(4) or 10(5) PFU), the behavior of wt RPV and RPV delta 35 became indistinguishable and the overall LD50 values were similar. Intradermal infection of rabbits simultaneously, at separate sites, with RPV and RPV delta 35 showed no gross or microscopic differences between either primary skin lesions or viremic extension of each virus into the lungs. Therefore, this abundant secreted protein does not appear to play a major role in the virulence of the virus.