Membrane potential-dependent ATP production was measured in mitochondrial fractions of procyclic Trypanosoma brucei using a luciferase based assay. Mitochondria isolated under hypotonic conditions were able to produce ATP using succinate as substrate. The same was observed with mitochondria isolated under isotonic conditions, however, in this case a 6-7-fold higher amount of ATP was produced with glycerol-3-phosphate as substrate. Disruption of the outer membrane of isotonically prepared mitochondria lead to a selective loss of the glycerol-3 phosphate induced ATP production, indicating that glycerol-3-phosphate dehydrogenase is a soluble enzyme of the intermembrane space. Isolation of mitochondria under hypotonic conditions, therefore, results in disruption of the outer membrane, whereas in the organelles isolated under isotonic conditions both the membranes remain intact.