Arginine biosynthesis in eukaryotes is divided between the mitochondria and the cytosol. The anaerobic chytridiomycete Neocallimastix frontalis contains highly reduced, anaerobic modifications of mitochondria, the hydrogenosomes. Hydrogenosomes also occur in the microaerophilic flagellate Trichomonas vaginalis, which does not produce arginine but uses one of the mitochondrial enzymes, ornithine transcarbamoylase, in a cytosolic arginine dihydrolase pathway for ATP generation. EST sequencing and analysis of the hydrogenosomal proteome of N. frontalis provided evidence for two mitochondrial enzymes of arginine biosynthesis, carbamoylphosphate synthase and ornithine transcarbamoylase, while activities of the arginine dehydrolase pathway enzymes were not detectable in this fungus.