Hexamita inflata is an amitochondriate flagellated protozoon which inhabits O(2)-limited environments. With the aid of 1H NMR spectroscopy, analysis of the metabolic fluxes in H. inflata grown in complex media under limited O(2) was performed. Almost complete carbon recovery from maltose (the principle carbohydrate source in the medium) catabolism was calculated from the measured increase in concentration of ethanol, alanine, acetate and lactate (and estimated CO(2) production). Difference spectra and amino acid analysis also identified changes in concentration of metabolites belonging to the arginine dihydrolase (ADH) pathway. The enzymes of the ADH pathway were detected in extracts with the following activities (in nmoles min(-1) x (mg of protein) x (-1)): arginine deiminase, 3.30; catabolic ornithine carbamyltransferase (OCT), 1.3; anabolic OCT, 93.0; and carbamate kinase, 1829. The organism metabolized the ornithine produced from catabolic OCT activity to putrescine via ornithine decarboxylase (ODC). The polyamines, spermidine and spermine, were formed by the sequential addition of the aminopropyl group of decarboxylated S-adenosyl-L-methionine (SAM) by the respective polyamine synthases. In addition, asparaginase activity was confirmed in H. inflata, catalysing the deamination of asparagine generating aspartate and ammonia. This study also indicates that, as with other amitochondriate protozoa and some bacteria, the ADH pathway significantly contributes to the energy yield of the cell, particularly under O(2)-limited conditions.