Protozoan parasites lack de novo purine biosynthesis and require purine salvage from the host. Nucleoside hydrolases are involved in nucleoside salvage and are not found in mammals, making them protozoan-specific targets for inhibitor design. Several protozoan nucleoside hydrolase isozymes with distinct substrate specificities have been characterized. Novel substituted iminoribitols have been synthesized to resemble the transition state structure of the nonspecific inosine-uridine nucleoside hydrolase from Crithidia fasciculata (IU-nucleoside hydrolase). These inhibitors have been characterized for this enzyme and for a purine-specific nucleoside hydrolase (IAG-nucleoside hydrolase) from Trypanosoma brucei brucei. Inhibitors which provide nanomolar inhibition constants for IU-nucleoside hydrolase exhibit micromolar inhibition constants for the IAG-enzyme. For example, p-bromophenyliminoribitol inhibits the IU- and IAG-enzymes with dissociation constants of 28 nM and 190 microM, respectively. Substrate specificity, the action of transition state inhibitors and the pH-dependence of the kinetic constants establish that the catalytic mechanisms and transition state structures are fundamentally different for the IU- and IAG-isozymes. The finding is remarkable since these isozymes share significant homology at the catalytic sites and both use inosine as a preferred substrate. The specificity of the transition state analogues indicates that logically-designed transition state inhibitors are isozyme-specific, with (Km/Ki IU-nucleoside hydrolase)/(Km/Ki IAG-nucleoside hydrolase) values up to 39,000. The mechanism of the differential inhibition is based on the relative leaving group activation and ribosyl-oxocarbenium-forming abilities of these enzymes. In addition to providing isozyme-specific inhibitors, the novel molecules described here have diagnostic value for the nature of the transition states for N-ribohydrolase enzymes.