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Trichomonas vaginalis and Tritrichomonas foetus are pathogens that parasitise, respectively, human and bovine urogenital tracts causing disease. Using LC-MS, reference metabolomic profiles were obtained for both species and stable isotope labelling with D-[U-13C6] glucose was used to analyse central carbon metabolism. This facilitated a comparison of the metabolic pathways of T. vaginalis and T. foetus, extending earlier targeted biochemical studies. 43 metabolites, whose identities were confirmed by comparison of their retention times with authentic standards, occurred at more than 3-fold difference in peak intensity between T. vaginalis and T. foetus. 18 metabolites that were removed from or released into the medium during growth also showed more than 3-fold difference between the species. Major differences were observed in cysteine and methionine metabolism in which homocysteine, produced as a bi-product of trans-methylation, is catabolised by methionine γ-lyase in T. vaginalis but converted to cystathionine in T. foetus. Both species synthesise methylthioadenosine by an unusual mechanism, but it is not used as a substrate for methionine recycling. T. vaginalis also produces and exports high levels of S-methylcysteine, whereas only negligible levels were found in T. foetus which maintains significantly higher intracellular levels of cysteine. 13C-labeling confirmed that both cysteine and S-methylcysteine are synthesised by T. vaginalis; S-methylcysteine can be generated by recombinant T. vaginalis cysteine synthase using phosphoserine and methanethiol. T. foetus contained higher levels of ornithine and citrulline than T. vaginalis and exported increased levels of putrescine, suggesting greater flux through the arginine dihydrolase pathway. T. vaginalis produced and exported hydroxy acid derivatives of certain amino acids, particularly 2-hydroxyisocaproic acid derived from leucine, whereas negligible levels of these metabolites occurred in T. foetus.