To assess the expression and physiological role of the mitochondrial NAD+-independent lactate dehydrogenase (iLDH) in Euglena gracilis, cells were grown with different carbon sources, and the d- and l-iLDH activities and several key metabolic intermediates were examined. iLDH activity was significant throughout the growth period, increasing by three- to fourfold from latency to the stationary phase. Intracellular levels of d- and l-lactate were high (5–40 mm) from the start of the culture and increased (20–80 mm) when the stationary phase was entered. All external carbon sources were actively consumed, reaching a minimum upon entering the stationary phase, when degradation of paramylon started. The level of ATP was essentially unchanged under all experimental conditions. Oxalate, an inhibitor of iLDH, strongly inhibited oligomycin-sensitive respiration and growth, whereas rotenone, an inhibitor of respiratory complex I, only slightly affected these parameters in lactate-grown cells. Isolated mitochondria exhibited external NADH-supported respiration, which was sensitive to rotenone and flavone, and an inability to oxidize pyruvate. Addition of cytosol, NADH and pyruvate to mitochondria incubated with rotenone and flavone prompted significant O2 uptake, which was blocked by oxalate. The data suggested that iLDH expression in Euglena is independent of substrate availability and that iLDHs play a key role in the transfer of reducing equivalents from the cytosol to the respiratory chain (lactate shuttle).