Several lines of evidence are presented that an entity is present in mitochondria which converts lysine and carbamylphosphate to homocitrulline, distinct from the ornithine transcarbamylase (OTC): lack of inhibition by lysine of OTC with ornithine as substrate at the pH of the mitochondrial matrix, a Km for lysine of 6.3 mmol/l in the crude mitochondrial extract, versus a Km of 55.3 mmol/l after partial purification of OTC, decrease of the specific activity of OTC with lysine as substrate and increase of the specific activity with ornithine as substrate after heat denaturation, and separation of the activities for ornithine and lysine after isoelectric focusing. This would explain the excretion of homocritrulline in the HHH-syndrome, saccharopinuria, citrullinemia and hyperlysinemia. The fact that homocitrulline excretion is not observed in OTC deficiency suggests that the activity responsible for homocitrulline excretion is closely related to OTC.