Metronidazole (Mz)-resistant Giardia and Trichomonas were inhibited by 1 of 30 new 5-nitroimidazole drugs.Another five drugs were effective against some but not all of the Mz-resistant parasites. This study provides the incentive for the continued design of 5-nitroimidazole drugs to bypass cross-resistance among established 5-nitromidazole antiparasitic drugs.Metronidazole (Mz) and a related 5-nitroimidazole, tinidazole, are the only drugs recommended for the treatment of trichomoniasis and are the most-prescribed drugs for the treatment of giardiasis. However, clinical resistance to these drugs has been well documented; and in the event of overt clinical resistance to Mz in trichomonads, there is no alternative for treatment, when one keeps in mind the documented crossresistance between the currently used 5-nitroimidazole drugs and their worldwide availability (7,8,18,23). Some success has been obtained with quinacrine and albendazole in combination with Mz in cases of giardiasis treatment failures (23). On the positive side, a great deal of flexibility is offered by the side chains attached to the imidazole ring structure that bear the all important nitro group (17).The mechanisms of Mz resistance in Giardia and Trichomonas have been well studied in laboratory-induced resistance (18). It occurs by down-regulation of pathways, especially the enzyme pyruvate:ferredoxin oxidoreductase (PFOR) and ferredoxin (Fd) pathway, that activate Mz to its toxic radical state. The PFOR-Fd couple has an electron potential sufficiently low to activate Mz, while no such electron couple is present in the mammalian host (9). In the laboratory we see a threefold down-regulation of PFOR activity in Mz-resistant (Mz r ) Giardia duodenalis (14), and in highly Mz r Trichomonas vaginalis the activity of the hydrogenosome organelle is downregulated such that there is no detectable PFOR or Fd expression (4,11,18). Thus, Mz is not activated to its toxic radical state in these cells. On the other hand, it is well documented that clinically Mz r T. vaginalis strains do not have down-regulated hydrogenosomes, and the mechanism of Mz resistance in these cells is not understood (8).Previously, we showed that some 5-nitroimidazole derivatives were significantly more effective antiprotozoal agents (based on in vitro molar drug concentrations) than Mz against Mz-susceptible (Mz s ) parasites but were not as effective against Mz r parasites (17). Given the impetus for the development of 5-nitroimidazole drugs that vary markedly in their efficacies (both positively and negatively), we tested 30 new 5-nitroimidazoles in our anaerobic drug susceptibility screening assay (16) for their efficacies against T. vaginalis and G. duodenalis, with the focus on laboratory-derived Mz r (Mz rl ) lines and clinical isolates derived from patients with treatment failures.Parasites were cultured axenically in anaerobic TYI-S-33 (6), which was modified as described previously (16 All new 5-nitroimidazole compounds (Fig. 1) were identified by spectral data, purified b...