Administration of recombinant rat gamma-interferon to rats conferred complete protection against an otherwise lethal intraperitoneal pseudorabies virus (PRV) infection. The primary target cell of the virus has been identified as the serosal cell of the peritoneum. Histologic examination showed that after infection of the underlying adventitia, the virus replicates in the myenteric and submucosal plexuses of the gastrointestinal tract; this is followed by centripetal spread to the autonomous and central nervous system. In recombinant rat gamma-interferon-treated rats, viral antigen was absent in the primary target cells and was not detected in any other organ. In interferon-treated cultures of peritoneal fibroblasts, which represent another primary target cell population in vivo, complete inhibition of PRV replication was observed. The peritoneal macrophage is not susceptible to PRV, as was shown by coculture and immunocytochemical studies. Peritoneal cells from gamma-interferon-treated rats showed enhanced major histocompatibility class II antigen expression and extrinsic antiviral activity in PRV-susceptible rat embryo fibroblasts. The results presented in this study indicate that protection by the lymphokine is likely to be based on direct inhibition of viral replication in serosal cells.