Pretreatment of tissue culture cells with alpha interferon (IFN-alpha) inhibits the transcription of herpes simplex virus type 1 (HSV-1) immediate-early (IE) genes, an effect which has been attributed to reduced transactivation of IE promoters by the virion protein VP16. Our previous demonstration that IFN-alpha inhibited the replication of the HSV-1 mutant in1814, which has a mutated VP16 unable to activate IE transcription, appeared to be incompatible with IFN-alpha having an effect on VP16 action (D. R. S. Jamieson, L. H. Robinson, J. I. Daksis, M. J. Nicholl, and C. M. Preston, J. Gen. Virol. 76:1417-1431, 1995). To investigate this observation further, cells were infected with a derivative of in1814 containing the lacZ gene controlled by the human cytomegalovirus IE promoter. The accumulation of HSV-1 IE RNA species was inhibited by IFN-alpha in these cells to the same extent as in cells infected with a virus rescued at the VP16 locus, and production of lacZ-specific RNA was also reduced, demonstrating that IFN-alpha can inhibit expression from a heterologous promoter that is not responsive to VP16. To provide a means of investigating the activity of VP16 on IE promoters not located in the HSV-1 genome, cell lines containing the neomycin phosphotransferase gene controlled by the HSV-1 IE ICPO promoter were constructed. Activation of the IE promoter by VP16 was not inhibited when the ICPO promoter was resident in the cell, demonstrating that VP16 function was unaffected by pretreatment of cells with IFN-alpha. The results suggest that IFN-alpha prevents the onset of IE transcription from the HSV-1 genome through a general mechanism rather than by having an effect specific to HSV-1 IE promoters.