Derivatives of 5-nitroimidazole, such as metronidazole or tinidazole, are the drugs of choice for treatment of sexually transmitted infections of humans caused by the parasitic protist Trichomonas vaginalis. These drugs with selective activity against anaerobic and microaerophilic microorganisms enter the trichomonad cell and accumulate in hydrogenosomes, where their antimicrobial properties are activated. In this chapter we discuss metabolic pathways of hydrogenosomes involved in metronidazole activation. We also summarize present knowledge on the development and biochemical mechanisms of metronidazole resistance in T. vaginalis and the related cattle parasite Tritrichomonas foetus. Implications of data from the T. vaginalis genome project suggesting the presence of novel mechanisms of drug resistance are also considered.