The E3L gene is essential for pathogenesis in vaccinia virus. The E3L gene product consists of an N-terminal Z alpha domain and a C-terminal double-stranded RNA (dsRNA) binding domain; the left-handed Z-DNA-binding activity of the Z alpha domain of E3L is required for viral pathogenicity in mice. E3L is highly conserved among poxviruses, including the smallpox virus, and it is likely that the orthologous Z alpha domains play similar roles. To better understand the biological function of E3L proteins, we have investigated the Z-DNA-binding behavior of five representative Z alpha domains from poxviruses. Using surface plasmon resonance (SPR), we have demonstrated that these viral Z alpha domains bind Z-DNA tightly. Ability of Z alpha(E3L) converting B-DNA to Z-DNA was measured by circular dichroism (CD). The extents to which these Z alphas can stabilize Z-DNA vary considerably. Mutational studies demonstrate that residues in the loop of the beta-wing play an important role in this stabilization. Notably the Z alpha domain of vaccinia E3L acquires ability to convert B-DNA to Z-DNA by mutating amino acid residues in this region. Differences in the host cells of the various poxviruses may require different abilities to stabilize Z-DNA; this may be reflected in the observed differences in behavior in these Zalpha proteins.