Medium of untreated human FS-4 foreskin fibroblasts contained a factor which, upon the addition of exogenous tumor necrosis factor (TNF), inhibited encephalomyocarditis virus replication when neither medium alone nor TNF alone were effective. This antiviral activity was abolished by a monoclonal antibody to human interferon (IFN)-beta, suggesting that the active component in the medium from untreated FS-4 cells was IFN-beta, present at subeffective concentrations. In addition, we show that untreated FS-4 cells contain IFN-beta mRNA, demonstrable by the highly sensitive polymerase chain reaction after reverse transcription. Treatment of FS-4 cells with TNF produced an approximately 16-fold increase in the steady-state level of IFN-beta mRNA. Our results support the conclusion that autocrine IFN-beta is secreted by untreated normal fibroblasts and that TNF can enhance the production of autocrine IFN-beta by increasing the level of IFN-beta mRNA. Our study also demonstrates that subeffective concentrations of autocrine IFN-beta, which escape detection in conventional assays, are sufficient to produce a strong synergistic action with TNF.