Mitochondrial dysfunction reduces aerobic energy production and results in symptoms from various tissues, depending on metabolic demands. Mitochondrial adenosine triphosphate (ATP) is essential for sperm motility. Sperm motility was investigated in a patient with a mitochondrial disease caused by reduced activity of the mitochondrial enzyme complexes I and IV, and in two control subjects. Spermatozoa were cultured in media containing various energy substrates. Motility was judged by light microscopy, and ultrastructure by transmission electron microscopy. In the patient with mitochondrial disease, 12% of the spermatozoa were motile in the medium containing only glucose. There was a three-fold increase in motile spermatozoa when pyruvate and succinate were present together with glucose. In contrast, the spermatozoa of both control subjects had best motility in the presence of substrates for complex I, and no further increase was observed when succinate was added. Glucose and pyruvate enter the respiratory chain at complex I, and succinate at complex II. Electron microscopy of spermatozoa from the patient with mitochondrial disease revealed mitochondria with increased matrix, thickening of membranes, parallelization of cristae and lipid inclusions, which are characteristic findings in mitochondrial disorders. Abnormal mitochondria were also found in a spermatid, suggesting that the ultrastructural changes of mitochondria are primary rather than secondary to degeneration of the spermatozoa. The results indicate that mitochondrial dysfunction causes reduced sperm motility in some men.