The rabbitpox virus (RPV) B5R protein, synthesized late in infection, is found as a 45-kDa membrane-associated protein of the envelope of infectious extracellular enveloped virus (EEV) and as a 38-kDa protein secreted from the cell by a process independent of morphogenesis. The protein is not found associated with intracellular mature virus (IMV). Deletion of the gene attenuates the virus (RPV delta B5R) in animals (mice and rabbits), has relatively little effect on formation of IMV, prevents EEV formation in some but not all cells, and leads to a reduced host range. Analysis of the sequence of the protein suggests relatedness to factor H of the complement cascade. Collectively, these observations suggest that attenuation of the virus in vivo could be linked to an inhibition of the inflammatory response, a deficiency in growth, or both. In this report we have analyzed the behavior of RPV delta B5R in infected mice and rabbits and conclude that attenuation of the mutant virus likely results from simple failure to grow within the infected animal and that the inflammatory response probably contributes little to the observed attenuation.