Foot-and-mouth disease virus protease 3C is essential for the processing of the viral precursor polyprotein. It is shown here to also inhibit gene expression in baby hamster kidney cells after transient expression from transfected cDNA fragments. Protease 3C could not be detected by indirect immunofluorescence in contrast to other cDNA-encoded virus proteins, but protein synthesized de novo 16 hr after transfection could be detected by radioimmunoprecipitation. The cellular translation apparatus was, therefore, not inhibited. The enzyme, although produced as part of a fusion protein, was in size indistinguishable from that found in virus-infected cells. This suggested that the enzyme was released by autocatalysis from the recombinant fusion protein and from viral precursor protein in a similar manner. Transcription of protease 3C-encoding cDNA fragments as well as that of cotransfected fragments, which do not encode protease 3C, was inhibited as determined by hybridization assays. The shut off of transcription which was one of the cytopathic effects observed in virus-infected cells therefore correlates with the production of transactive protease 3C. The inhibitory molecular mechanism may involve truncation of the nuclear protein histone H3 at its N-terminus since this protein was found similarly truncated in virus-infected cells and after transfer of 3C-encoding cDNA fragments.