The effect of inhibitors of mRNA synthesis (actinomycin D and alpha-amanitin) and DNA replication (mitomycin C and ethidium bromide) on Machupo virus reproduction was studied. Actinomycin D (1.0-4.0 micrograms/ml) and alpha-amanitin (10.0 micrograms/ml) added immediately after adsorption reduced the infectious virus titre by 2 lg or more. Actinomycin D inhibited virus reproduction not only at the early but also at the later stages of infection. Mitomycin C and ethidium bromide did not inhibit Machupo virus reproduction. The cells treated with actinomycin D at early and late stages of infection were found to contain immunoprecipitable virus-specific proteins 78, 64, and 37 KD described previously. The total amount of virus-specific proteins in the inhibitor-treated cells was reduced 3.7 and 2,6-fold after addition of actinomycin at 0 and 12 h postinfection, respectively, as compared with untreated cells. The mechanism of action of actinomycin D on the Machupo virus reproduction is discussed.