Decreased activity of complex I (NAD:ubiquinone oxidoreductase) is the most frequent biochemical finding associated with mutation at the base pair 3243 of the mitochondrial DNA. The mutation has been previously shown to lead to a defective translation. We hypothesized that due to an imperfect assembly of complex I subunits the substrate affinity of this enzyme may be lowered and this may be counteracted by increasing the mitochondrial NAD+NADH concentration. Therefore, we studied the effect and mechanism of action of nicotinamide treatment in a MELAS patient with the base pair 3243 mutation. Nicotinamide treatment was initiated after his first stroke-like episode. The blood NAD concentration (representing the intracellular concentration in erythrocytes) increased linearly being 24-fold at 6 weeks of treatment. Blood lactate and pyruvate concentration decreased by 50% within three days and 24 h urine lactate content within 2 weeks and we observed a clinical improvement together with a decrease in the lesion volume in magnetic resonance imaging within the first month. The cellular NAD increase upon nicotinamide administration was probably universal, because it occurred in a time and dose-dependent manner in cultured fibroblasts from both the patient and the controls. Alleviation of the lactate accumulation during the nicotinamide treatment suggests that an increase in the cellular NAD+NADH concentration leads to enhancement of the oxidation of reducing equivalents. However, the Km of complex I for NADH in skeletal muscle from the patient was similar to that of controls. This may indicate that physiologically mitochondrial complex I operates at non-saturating substrate concentration, and this may explain the effect of nicotinamide treatment.