Family 18 chitinases play key roles in a range of pathogenic organisms and are overexpressed in the asthmatic lung. By screening a library of marketed drug molecules, we have identified methylxanthine derivatives as possible inhibitor leads. These derivatives, theophylline, caffeine, and pentoxifylline, are used therapeutically as antiinflammatory agents, with pleiotropic mechanisms of action. Here it is shown that they are also competitive inhibitors against a fungal family 18 chitinase, with pentoxifylline being the most potent (K(i) of 37 microM). Crystallographic analysis of chitinase-inhibitor complexes revealed specific interactions with the active site, mimicking the reaction intermediate analog, allosamidin. Mutagenesis identified the key active site residues, conserved in mammalian chitinases, which contribute to inhibitor affinity. Enzyme assays also revealed that these methylxanthines are active against human chitinases.