The effects of ruminal concentrations of CO2 and O2 on glucose-stimulated and endogenous fermentation of the rumen ciliate Eudiplodinium maggii were investigated. The principal metabolic products were butyrate, acetate, lactate, propionate, H-2, and CO2. C-13 NMR spectroscopy revealed glycerol to be an important, but previously unidentified, fermentation product of this organism. Glucose uptake and metabolite formation rates were influenced by the headspace composition during protozoal incubations. Glucose uptake was most rapid in the presence of low O2 in N2 (1-3-mu-M O2 dissolved in the protozoal suspension). Pathways located in the hydrogenosomes were O2 sensitive, and low O2 concentrations resulted in lowered acetate, H-2, and CO2 formation. The presence of high CO2 (65% gaseous headspace by volume) resulted in elevated acetate and butyrate formation; fumarate and propionate were similarly found to accumulate at higher concentration than previously detected in the supernatants. Results suggest that under conditions similar to those prevailing in the rumen (i.e., high CO2), Eu. maggii produces higher levels of important ruminal volatile fatty acids, and thus its relative contribution to rumen metabolism may have been underestimated.