Deletion or truncation of NS1, the principal 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 wild-type 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 insufficient per se 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.