The Shope fibroma virus (SFV) DNA topoisomerase gene has been identified and mapped to the BamHI D fragment near the midpoint of the genome. The DNA sequence of the SFV BamHI S fragment together with the contiguous BamHI-ClaI subfragment of BamHI D which encompasses the topoisomerase gene and two flanking genes has been determined and analyzed. Both the SFV DNA topoisomerase and the two flanking genes are closely related in terms of sequence and spatial organization to the homologous sequences from the midpoint of the vaccinia virus genome, indicating that these proteins are conserved not only in their sequence but also by position within the poxvirus genome. To confirm the assignment of the SFV gene, the putative SFV DNA topoisomerase has been expressed as an active fusion protein in Escherichia coli and this system should be useful in the analysis of topoisomerase function following the introduction of targeted mutations into the topoisomerase gene. The results of this work shed further light on the evolutionary relationship of the different poxvirus genera and indicate that central unique regions of the poxvirus genomes contain many of the essential viral genes and are thus highly conserved.