Since the macrolide polyene antibiotics, amphotericin B (AmB) and its methyl ester (AmBME), augment interferon production by polyriboinosinic:polyribocytidylic acid [poly(I):poly(C)] in vitro, experiments were undertaken to determine how AmB and AmBME affect the antiviral activity of poly(I):poly(C) and interferon. AmBME increased the direct antiviral activity of poly(I):poly(C) 10(2)-to 10(4)-fold in L929, Flow 6000, and T98G cells. Viral replication, measured by either direct plaque formation or virus yield, was markedly reduced. Serum interferon levels in mice induced by poly(I):poly(C) were enhanced by concomitant treatment with AmB. However, the therapeutic effects of poly(I):poly(C) in encephalomyocarditis and Semliki Forest virus infections were not augmented by combined treatment with poly(I):poly(C) and AmB. In vitro, the antiviral effects of exogenous interferon were markedly inhibited by AmB and AmBME. This inhibition may have contributed to the adverse effects of the macrolide polyenes in encephalomyocarditis and Semliki Forest virus infections in vivo. These findings further substantiate the effectiveness of macrolide polyenes in augmenting cellular penetration of macromolecules. However, therapeutic application may be limited by the complex interactions which occur between compounds administered in vivo.