An enzyme immunoassay (EIA) to determine 50% inhibitory concentrations of ribavirin which suppresses Semliki Forest virus (SFV) multiplication in L-cells is described. Inhibition of SFV replication by ribavirin was measured by detection of viral glycoprotein, on the surface of infected L-cell monolayers, with a horseradish peroxidase labeled monoclonal antibody with specificity for the E2 glycoprotein of SFV. The concentration of ribavirin in culture fluid associated with 50% reduction of control absorbance values was defined as the 50% inhibitory concentration (IC50). The IC50 of ribavirin measured with EIA was mainly influenced by the multiplicity of infection (MOI). At MOI values of 3, 6 and 12 reduction of absorbance values was already obvious at 4.5 h of infection. Furthermore reduction of absorbance values correlated with inhibition of virus production as determined by plaque titration of culture fluids. When the EIA was used for the determination of active ribavirin in serum from treated animals the drug was detectable 1 h after intravenous administration of 4 mg of ribavirin to a concentration of 8 micrograms per ml serum. The results indicate that this simple EIA is suitable for the monitoring of active antiviral drugs in body fluids.