cDNA sequencing revealed that chick Mx protein consists of 705 amino acids. Its 84 N-terminal amino acids show no significant sequence homology to other Mx proteins. They are followed by 514 residues that include a tripartite GTP binding consensus motif. This region shows 50-70% sequence identity to mammalian and duck Mx proteins. Sequences near the C terminus, including a leucine zipper motif, are also conserved, whereas the intervening 19 amino acids lack sequence similarity. This unique sequence corresponds to a highly variable region in mammalian Mx proteins, suggesting that it serves as a spacer between functional domains. Chick and mouse cells transiently transfected with cDNA expression constructs synthesized chick Mx protein at a level that could easily be detected with specific antibodies. Chick Mx protein in such cells was mainly cytoplasmic and had a granular appearance. Permanently transfected cell lines expressing high levels of chick Mx protein could not be established, suggesting low metabolic stability of chick Mx protein or incompatibility with cell proliferation. The antiviral activity of chick Mx protein was tested at the single-cell level using immunofluorescence techniques. Transfected cells expressing chick Mx protein showed no enhanced resistance to influenza A virus, vesicular stomatitis virus, Thogoto virus, or Sendai virus. Thus, chick Mx joins the list of Mx proteins without recognized antiviral activity, supporting the concept that Mx proteins serve unrelated functions.