I ntravenous injection of polyinosinic acid/polycytidylic acid [(poly rI)·(poly rC)] offered significant protection against intranasal challenge of young mice with vesicular stomatitis virus (VSV). Optimal protection was obtained when a single dose was administered 2 hr before virus challenge, but repeated doses were effective when started as late as 3 days after virus challenge. The therapeutic ratio or ratio of maximum tolerated dose to minimum effective dose for a single intravenous injection of (poly rI)·(poly rC) 2 hr before virus inoculation was ≥8 mg/kg:0.004 mg/kg or ≥200.Dose-response curves for interferon production and antiviral protection by (poly rI)·(poly rC) were closely parallel. Equivalent doses of poly rI or poly rC alone did not exert any interferon-inducing capacity or protective effect on intranasal VSV challenge. Several factors, which are known to potentiate or antagonize interferon production, increased or decreased the interferon-inducing capacity and antiviral protection of either (poly rI)·(poly rC) or maleic acid/divinyl ether copolymer (MA/DVE) in parallel. Interferon production and antiviral protection by MA/DVE were enhanced by arginine but abolished by prior treatment with MA/DVE; DEAE-dextran (intraperitoneally), kinetin riboside and isopentenyladenosine, and prior injection of endotoxin reduced both interferon production and antiviral protection by (poly rI)·(poly rC).Treatment with exogenous interferon in amounts which closely mimicked the levels of circulating interferon produced endogenously by an effective dose of (poly rI)·(poly rC) gave protection against intranasal VSV which was identical with that dose of (poly rI)·(poly rC). This strongly suggests that interferon production accounts for the whole protective effect of (poly rI)·(poly rC) in the intranasal VSV assay.