Aims: Medicinal plants are increasingly being projected as suitable alternative sources of antiviral agents. The development of a suitable in vitro pharmacodynamic screening technique could contribute to rapid identification of potential bioactive plants and also to the standardization and/or pharmacokinetic-pharmacodynamic profiling of the bioactive components. Methods and Results: Recombinant viral vectors ( lentiviral, retroviral and adenoviral) transferring the firefly luciferase gene were constructed and the inhibition of viral vector infectivity by various concentrations of plant extracts was evaluated in HeLa or Hep2 cells by measuring the changes in luciferase activity. Cytotoxicity of the extracts was evaluated in parallel on HeLa or Hep2 cells stably expressing luciferase. Amongst the 15 extracts screened, only the methanol ( ME) and the ethyl acetate ( ET) fractions of the lichen, Ramalina farinacea specifically reduced lentiviral and adenoviral infectivity in a dose-dependent manner. Further, chromatographic fractionation of ET into four fractions (ET1-ET4) revealed only ET4 to be selectively antiviral with an IC50 in the 20 mu g ml(-1) range. Preliminary mechanistic studies based on the addition of the extracts at different time points in the viral infection cycle ( kinetic studies) revealed that the inhibitory activity was highest if extract and vectors were preincubated prior to infection, suggesting that early steps in the lentiviral or adenoviral replication cycle could be the major target of ET4. Inhibition of wild-type HIV-1 was also observed at a 10-fold lower concentration of the extract. Conclusions: The vector-based assay is a suitable in vitro pharmacodynamic evaluation technique for antiviral medicinal plants. The technique has successfully demonstrated the presence of antiviral principles in R. farinacea. Significance and Impact of Study: Potential anti-HIV medicinal plants could rapidly be evaluated with the reported vector-based technique. The lichen, R. farinacea could represent a lead source of antiviral substances and is thus worthy of further studies.