The inhibitory activity of caffeine, a pharmacologically active compound, on Junin virus (JV) multiplication in Vero cells was evaluated by a virus yield inhibition assay. The compound achieved a dose-dependent inhibition of virus production at concentrations not affecting cell viability. The 50% inhibitory concentration (IC50) and the 90% inhibitory concentration (IC90) were 9.0 mM and 10.8 mM, respectively. From time of addition experiments, it can be concluded that caffeine inhibited an early stage in the replicative cycle of JV occuring before 4 h of infection. Extracellular and cell-associated virus yields were reduced to the same extent. The addition of caffeine after several cycles of infection for a very short treatment period did not significantly affect the formation of JV infectious particles. The expression of viral proteins in the cytoplasm and the membrane of infected cells was highly reduced in the presence of caffeine, as revealed by immunofluorescence staining, confirming that caffeine predominantly exerted its inhibitory action early in the infection of Vero cells with JV.