Deletion or truncation of NS1, the principal interferon (IFN) antagonist of influenza viruses, leads to increased IFN induction during influenza virus infection. We have studied activation of the IFN induction cascade by both wildtype and NS1-defective viruses at the single-cell level using a cell line expressing GFP under the control of the IFN-β promoter and by examining MxA expression. The IFN-β promoter was not activated in all infected cells, even during NS1-defective virus infections. Loss of NS1 expression is therefore by itself insufficient to induce IFN in an infected cell, and factors besides NS1 expression status must dictate whether the IFN response is activated. The IFN response was efficiently stimulated in these cells following infection with other viruses; the differential IFN response we observe with influenza viruses is therefore not cell-specific, but is likely due to differences in the nature of the infecting virus particles and their subsequent replication.