The effect of hyaluronidase on cell response to poly(rI) . poly(rC) [poly(IC)] was investigated in rabbit kidney (RK) cells. For this purpose, bovine testicular and Staphylococcal hyaluronidase preparations at various degrees of purity were used. These enzyme preparations were employed at the maximal nontoxic dose for 2 hours before poly(IC) treatment. This enzymatic pretreatment of the cells strongly inhibited the antiviral activity of poly(IC), determined by using both Herpes simplex virus type-1 and vesicular stomatitis virus. It also remarkably reduced the poly(IC)-induced interferon production. This later effect could account for the diminished antiviral activity of poly(IC) in the hyaluronidase-treated cells.