Scytalone dehydratase (SD) is a molecular target of inhibitor design efforts aimed at protecting rice plants from the fungal disease caused by Magnaporthe grisea. As determined from X-ray diffraction data of an SD-inhibitor complex [Lundqvist et al. (1994) Structure (London) 2, 937-944], there is an extended hydrogen-bonding network between protein side chains, the inhibitor, and two bound water molecules. From models of SD complexed to quinazoline and benztriazine inhibitors, a new class of potent SD inhibitors involving the displacement of an active-site water molecule were designed. We were able to increase inhibitory potency by synthesizing compounds with a nitrile functionality displayed into the space occupied by one of the crystallographic water molecules. Sixteen inhibitors are compared. The net conversion of potent quinazoline and benztriazine inhibitors to cyanoquinolines and cyanocinnolines increased binding potency 2-20-fold. Replacement of the nitrile with a hydrogen atom lowered binding affinity 100-30,000-fold. X-ray crystallographic data at 1.65 A resolution on a SD-inhibitor complex confirmed that the nitrile functionality displaced the water molecule as intended and that a favorable orientation was created with tyrosines 30 and 50 which had been part of the hydrogen-bonding network with the water molecule. Additional data on inhibitors presented herein reveals the importance of two hydrogen-bonding networks toward inhibitory potency: one between Asn131 and an appropriately positioned inhibitor heteroatom and one between a bound water molecule and a second inhibitor heteroatom.