Virtual screening of compound libraries is an alternative and complementary approach to high-throughput screening in the lead discovery process. A new strategy is described to search for possible leads of human carbonic anhydrase II, applying a protocol of several consecutive hierarchical filters involving a preselection based on functional group requirements and fast pharmacophore matching. A suitable pharmacophore is derived by a sophisticated "hot spot" analysis of the binding site to detect regions favorable for protein-ligand interactions. In subsequent steps, molecular similarity with known reference ligands is used to rerank the hits from the pharmacophore matching. Finally the best scored candidates are docked flexibly into the protein binding pocket. After examination of the affinity predictions, 13 compounds were selected for experimental testing. Of these 13, three could be shown to be subnanomolar, one is nanomolar, while a further seven are micromolar inhibitors. The binding mode of two hits could be confirmed by crystal structure analysis. The novelty of the discovered leads is best supported by the fact that a search in the patent literature showed the newly discovered subnanomolar compounds to comprise scaffolds not yet covered by existing patents.