Neomycin inhibits the production of interferon-beta (IFN-beta) in human fibroblast cells in response to Sendai virus or to poly(rI).poly(rC) in a concentration-dependent manner, and to the greatest extent effective when added prior to or up to 2 h after induction. This inhibitory effect is negated when the protein kinase C activator, SC-9, is present during IFN-beta production in response to poly(rI).poly(rC), but not in response to Sendai virus. These results suggest that in human cells both virus and poly(rI).poly(rC) utilize an early neomycin-sensitive signal transduction step for the production of IFN-beta; because neomycin binds specific phosphatidylinositol phosphates, both of these inducers very likely require hydrolysis of these phosphates.