Poxviruses encode a broad range of proteins that interfere with host immune functions such as soluble versions of cytokine receptors. Soluble virus tumour necrosis factor receptors (vTNFRs) were described originally in myxoma and Shope fibroma viruses. Cowpox virus (CPV) encodes three vTNFRs (CrmB, CrmC and CrmD). The genes equivalent to CrmB and CrmC in vaccinia virus (VV) Copenhagen are mutated and are named B28R/C22L and A53R, respectively. CrmD was identified recently in CPV and ectromelia virus but the gene is absent in VV Copenhagen. We have tested for expression of soluble binding activity for human TNF in cultures infected with 18 orthopoxviruses and have found that TNFRs are mostly absent but are produced by VV strains Lister, USSR and Evans, by the CPV elephantpox and by camelpox virus. Interestingly, we also found TNFR activity on the surface of cells infected with VV Lister, USSR and Evans. Sequence analysis of the relevant regions in VV Lister identified an intact A53R gene and an inactive B28R gene. Expression of VV Lister A53R in baculovirus and VV Western Reserve demonstrated that gene A53R encodes an active soluble vTNFR of 22 kDa. Expression and characterization of recombinant vTNFRs from VV Lister (A53R) and CPV (CrmB and CrmC) showed a similar binding specificity, with each receptor binding TNF from man, mouse and rat, but not human lymphotoxin-alpha. Lastly, the VV Lister and CPV vTNFRs bind human TNF with high affinity and prevent the binding of TNF to cellular receptors.