A simple in situ model of alamethicin-permeabilized isolated rat liver mitochondria was used to investigate the channeling of NADH between mitochondrial malate dehydrogenase (MDH) and NADH:ubiquinone oxidoreductase (complex 1). Alamethicin-induced pores in the mitochondrial inner membrane allow effective transport of low molecular mass components such as NAD(+)/NADH but not soluble proteins. Permeabilized mitochondria demonstrate high rates of respiration in the presence of malate/glutamate and NAD(+) due to coupled reaction between MDH and complex I. In the presence of pyruvate and lactate dehydrogenase, an extramitochondrial competitive NADH utilizing system, respiration of permeabilized mitochondria with malate/glutamate and NAD(+) was completely abolished. These data are in agreement with the free diffusion of NADH and do not support the suggestion of direct channeling of NADH from MDH to complex I. (C) 2004 Elsevier Inc. All rights reserved.