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The life cycle of Trypanosoma brucei brucei involves a series of differentiation steps characterized by marked changes in mitochondrial development and function. The bloodstream forms of this parasite completely lack cytochromes and have not been considered to have any Krebs cycle function. It has been suggested that glycolysis is the sole source of ATP in all bloodstream forms. However, earlier results indicated that in the mitochondria of the transitional intermediate/short stumpy bloodstream forms, the biochemical pathways are present that could allow intra-mitochondrial production of ATP. Using a high mannitol buffer to enhance permeability, we confirm previous observations showing that transitional forms maintain motility and respiratory activity with 2-oxoglutarate as the sole substrate. Using a luminometer to measure intracellular ATP levels via the luciferinlluciferase chemiluminescence assay, we show that these same transitional forms, but not long slender forms, maintain high levels of intracellular ATP in the presence of 2-oxoglutarate. Further, in the presence of bongkrekic acid, an inhibitor of the mitochondrial adenine nucleotide translocase, ATP levels are reduced with subsequent death and lysis of the cells when 2-oxoglutarate, but not glucose, is used as sole substrate. These data are direct evidence of ATP production by transitional bloodstream form mitochondria.Trypanosoma brucei brucei is of medical and economic importance as the agent of human African sleeping sickness and nagana in cattle. The life cycle of the parasite in the mammalian host begins with metacyclic forms injected with the bite of the tsetse fly vector. These metacyclics subsequently transform into the rapidly dividing long slender forms which in turn give rise to intermediate and short stumpy forms, stages suggested to be pre-adapted to survive in the vector if ingested with a blood meal. In this report we refer to the intermediate and short stumpy bloodstream forms as transitional bloodstream forms. Several aspects of the metabolism of bloodstream 7: brucei brucei are known to be unique and have been identified as targets for chemotherapy (Fairlamb, 1989;Clarkson and Brohn, 1976). Of special interest is the mitochondrion which undergoes marked differentiation and development as the organism progresses through its life cycle (Vickerman, 1965). The procyclic or insect midgut form has cytochromes and Krebs cycle function whereas these are both reported to be absent in bloodstream form mitochondria which have high respiratory activity but a limited range of function (for review see Fairlamb and Opperdoes, 1976).Glycolysis is generally considered the sole source of ATP in all bloodstream forms. However, earlier work by Vickerman (1965) and Flynn and Bowman (1973) showed that populations with significant numbers of transitional bloodstream forms were able to maintain cell motility and oxygen utilization in the presence of 2-oxoglutarate, a Krebs cycle intermediate. In contrast, long slender fosms lysed in the absence of Correspo...