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Partially purified virus preparations from sporophores of Agaricus bisporusaffected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from healthy sporophores. Enzyme activity was dependent upon the presence of Mg2+and the four nucleoside triphosphates and was insensitive to actinomycin D, a-amanitin, and rifampin. The 3H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 × 106and 1.4 × 106; they corresponded in size and hybridized to the major dsRNAs detected in the virus preparation by ethidium bromide staining. Cs2SO4equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 × 106and 1.4 × 106. The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs.