Phorbol myristate acetate (PMA) and the calcium ionophore, A23187, produce a dose-dependent inhibition of vesicular stomatitis virus (VSV) replication in FL cells, with maximum inhibition when cells are pretreated with 1 ng/ml of PMA or 0.5-mu-M A23187. No interferon (IFN) activity was detected in culture supernatants from cells treated with these agents, and addition of anti-human IFN antibodies did not prevent the inhibition of VSV replication that they caused. However, their antiviral activities were inhibited by actinomycin D or cyclohexamide treatment. Thus, the antiviral activities of PMA and A23187 involve de novo synthesis of protein but are not mediated through the production of IFN.