The flexible superimposition of biologically active ligands is a crucial step in ligand-based drug design. Here we present pharmACOphore, a new approach for pairwise as well as multiple flexible alignment of ligands based on ant colony optimization (ACO; Dorigo, M.; Stützle, T. Ant Colony Optimization; MIT Press: Cambridge, MA, USA, 2004). An empirical scoring function is used, which describes ligand similarity by minimizing the distance of pharmacophoric features. The scoring function was parametrized on pairwise alignments of ligand sets for four proteins from diverse protein families (cyclooxygenase-2, cyclin-dependent kinase 2, factor Xa and peroxisome proliferator-activated receptor γ). The derived parameters were assessed with respect to pose prediction performance on the independent FlexS data set ( Lemmen, C.; Lengauer, T.; Klebe, G. J. Med. Chem. 1998, 41, 4502 - 4520) in exhausting pairwise alignments. Additionally, multiple flexible alignment experiments were carried out for the pharmacologically relevant targets trypsin and poly (ADP-ribose) polymerase (PARP). The results obtained show that the new procedure provides a robust and efficient way for the pairwise as well as multiple flexible alignment of small molecules.