An immunofluorescent assay (IFA) for Pichinde virus (PCV), a member of the family Arenaviridae, was developed for antiviral drug assays against the virus. The assay was performed by adding fluorescein-labeled anti-PCV monoclonal antibody to virus-infected cells at 24 h after the initial infection and counting the infected cells with an epifluorescence microscope. The average 50% effective dose (ED50) for a series of nucleoside analogues tested against PCV using this IFA was: 2-beta-D-ribofuranosylselenazole-4-carboxamide (selenazofurin), less than 1.0 microgram/ml; 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin), 6.0 micrograms/ml; ammonium 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide- 5'-phosphate hydrate (ribavirin-5'-monophosphate), 15.8 micrograms/ml; ammonium 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide-5'-hemisuccinate (ribavirin-5'-hemisuccinate), 14.7 micrograms/ml; ammonium 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide-5'-(2,3- dimethyl)hemisuccinate [ribavirin-5'-(2,3-dimethyl)hemisuccinate], 213.5 micrograms/ml; 4-hydroxy-1-beta-D-ribofuranosyl-2-pyridone (3-deazauridine), 5.2 micrograms/ml; and (S)-9-(2,3-dihydroxypropyl)adenine, ([S]-DHPA), 471.0 micrograms/ml. In comparison, the ED50 of ribavirin using inhibition of marginal PCV-induced cytopathogenic effect after 12 days was 6.0 micrograms/ml and using plaque reduction after 5 days was 2.5 micrograms/ml, indicating that this IFA was of comparable sensitivity to these other tests.