We report on two families with Sengers syndrome and mutations in the acylglycerol kinase gene (AGK). In the first family, two brothers presented with vascular strokes, lactic acidosis, cardiomyopathy and cataracts, abnormal muscle cell histopathology and mitochondrial function. One proband had very abnormal mitochondria with citrate synthase crystals visible in electron micrographs, associated with markedly high citrate synthase activity. Exome sequencing was used to identify mutations in the AGK gene in the index patient. Targeted sequencing confirmed the same homozygous mutation (c.3G>A, p.M1I) in the brother. The second family had four affected members, of which we examined two. They also presented with similar clinical symptoms, but no strokes. Postmortem heart and skeletal muscle tissues showed low complex I, III and IV activities in the heart, but normal in the muscle. Skin fibroblasts showed elevated lactate/pyruvate ratios and low complex I+III activity. Targeted sequencing led to identification of a homozygous c.979A>T, p.K327* mutation. AGK is located in the mitochondria and phosphorylates monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid. Disruption of these signaling molecules affects the mitochondria's response to superoxide radicals, resulting in oxidative damage to mitochondrial DNA, lipids and proteins, and stimulation of cellular detoxification pathways. High levels of manganese superoxide dismutase protein were detected in all four affected individuals, consistent with increased free radical damage. Phosphatidic acid is also involved in the synthesis of phospholipids and its loss will result in changes to the lipid composition of the inner mitochondrial membrane. These effects manifest as cataract formation in the eye, respiratory chain dysfunction and cardiac hypertrophy in heart tissue. These two pedigrees confirm that mutation of AGK is responsible for the severe neonatal presentation of Sengers syndrome. The identification of citrate synthase precipitates by electron microscopy and the presence of vascular strokes in two siblings may expand the cellular and clinical phenotype of this disease.