The DNA sequence of a 2060-bp fragment from the left-hand end of the ectromelia (mousepox) virus genome was determined. Two genes were identified coding for 28 kDa (p28) and 16 kDa (p16) proteins, both of which are disrupted in vaccinia virus but conserved in variola (smallpox) virus. p16 contains an N-terminal hydrophobic region and may be a membrane or secreted protein. p28 contains a C3HC4 (RING) zinc finger motif that has been found in a large family of proteins involved mostly in transcription regulation. p28 was expressed in bacteria and shown to bind Zn in vitro. Disruption of the p28 gene had no appreciable effect on the multiplication of the virus in cell culture but abolished its lethality for susceptible mice. The p28- mutant virus replicated to significantly lower titers than the wild-type virus in different organs of infected mice. It is proposed that the p28 gene is an important determinant of orthopoxvirus pathogenicity, and its product may positively or negatively regulate expression of host or viral gene(s) involved in virus-host interaction.