Axenic culture amastigote-like forms of Trypanosoma cruzi, grown at 28 degrees C, reach a stationary phase after two generations, and differentiate to epimastigotes, which then resume growth. Axenic culture amastigotes readily ferment glucose to succinate and acetate, and do not excrete NH3; they have high activities of hexokinase and phosphoenolpyruvate carboxykinase, and very low citrate synthase activity; cytochrome o is absent, and cytochrome b-like is present at a very low level. Epimastigotes catabolize glucose and produce succinate and acetate at a considerably lower rate; they exhibit lower levels of hexokinase and carboxykinase, and much higher levels of citrate synthase and cytochromes o and b-like. They catabolize amino acids, as shown by excretion of NH3 to the medium. The results suggest that axenic culture amastigotes have an essentially glycolytic metabolism, and they acquire the ability to oxidize substrates such as amino acids only after differentiation to epimastigotes.