A new class of imidazoquinolines, including imiquimod, S-27609 and S-28463, has shown potent immunomodulating, antiviral and antitumor activities in animal models. Imiquimod is currently in phase III clinical trials for treating genital warts. These drugs were evaluated for immunomodulating activities in human PBMC, keratinocyte and fibroblast cultures and after topical application to mice. All three compounds induced a similar set of cytokines in human PBMC including interferon (IFN)-α, TNF-α, IL-1, IL-6 and IL-8. Concentrations of S-28463 ≥ 0.01 μg/ml were effective at inducing cytokines; whereas, imiquimod was only active at concentrations ≥ 0.5 μg/ml. In human keratinocytes neither imiquimod, S-27609 nor S-28463 induced release of IFN-α, IL-1α, IL-6, IL-8 or TNF-α. Human fibroblasts also failed to produce cytokines in response to these drugs. A topical formulation of imiquimod was also applied to the backs of mice, skin was taken, RNA was isolated and reverse transcriptase PCR was performed to examine induction of cytokine m-RNA. 5% imiquimod cream resulted in a 4 fold increase in m-RNA for IFN-α, but not TNF, IL-1α, IL-1β, IL-6, IL-8 or IFN-β. Similar results were seen when using a gel formulation of S-28463 which induced a 12 fold increase in comparison to the vehicle. These results suggest that topical application of the imidazoquinolines leads to the induction of IFN-α in skin and this may explain the effectiveness of imiquimod in the treatment of genital warts.