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The antiviral effects of 6-diazo-5-oxo-l-norleucine (L-DON) on the replication of human parainfluenza virus type 2 (HPIV-2), mumps and vesicular stomatitis viruses were studied. L-DON suppressed growth of these viruses and, in particular, HPIV-2 in four cell types. L-DON was not toxic to the cells at the active dose and did not significantly inhibit cellular macromolecular synthesis. The L-DON-sensitive step of HPIV-2 replication was considered to be relatively early. The NP, P and M proteins were, although at a low level, clearly detectable in HPIV-2-infected Vero cells treated with L-DON, whereas the HN and F proteins were scarcely detected by either immunostaining or immunoprecipitation, indicating that L-DON mainly decreased the amounts of viral glycoproteins. Furthermore, Northern blot hybridization showed that secondary transcription of virus RNA was also inhibited.