Cytoplasmic microinjection of murine Mx mRNA synthesized in vitro or nuclear microinjection of Mx cDNA under the control of a constitutive promoter into murine Mx- cells led to the accumulation of Mx protein in the nucleus and inhibited the replication of influenza virus but not of vesicular stomatitis virus (VSV). Similar results were also found with dog, rat, chicken, and monkey cells. A human lung fibroblast cell line (A549) was exceptional in that Mx protein was located predominantly in the cytoplasm and showed antiviral activity. Truncation of the 19 last residues of murine Mx protein almost completely abolished accumulation of Mx protein in the nucleus; however the activity against influenza virus was at least partially retained. The truncated region contains a segment rich in basic amino acids, similar to that reported for several nuclear location signals.