Postinduction repression of the beta-interferon gene is mediated through two positive regulatory domains. Virus induction of the human beta-interferon (beta-IFN) gene results in an increase in the rate of beta-IFN mRNA synthesis, followed by a rapid postinduction decrease. In this paper, we show that two beta-IFN promoter elements, positive regulatory domains I and II (PRDI and PRDII), which are required for virus induction of the beta-IFN gene are also required for the postinduction turnoff. Although protein synthesis is not necessary for activation, it is necessary for repression of these promoter elements. Examination of nuclear extracts from cells infected with virus reveals the presence of virus-inducible, cycloheximide-sensitive, DNA-binding activities that interact specifically with PRDI or PRDII. We propose that the postinduction repression of beta-IFN gene transcription involves virus-inducible repressors that either bind directly to the positive regulatory elements of the beta-IFN promoter or inactivate the positive regulatory factors bound to PRDI and PRDII. 