Two patients with neonatal onset of hyperornithinemia-hyperammonemia-homocitrullinuria syndrome were studied at 4 and 2 1/2 yr of age, respectively. The aim of the investigation was to assess the effect of supplementing citrulline, arginine, or ornithine (2 mmol/kg per day) while on a protein-restricted diet. The peroral supplementation was carried out during 2 wk for each amino acid. While ammonia in plasma was not increased the supply of citrulline or arginine led to a reduction of plasma glutamine compared to ornithine supplement or to no supplement (control period). Plasmatic ornithine was raised in all instances. Homocitrulline excretion was lower with all additions compared to the control period. Adding citrulline to the diet (in contrast to supplementing arginine) did not lower tubular lysine reabsorption. A lowered creatine excretion was found which could be normalized by arginine or citrulline. The data are compatible with a product inhibition of arginino-glycine transamidinase suggesting that the enzyme is not located in the mitochondrial matrix in man. Citrulline supplement combined with a protein-restricted diet appears to allow a normal development. The additional finding of a factor VII and X deficiency in one of the patient and reports in the literature of this association in two other patients with hyperornithinemia-hyperammonemia-homocitrullinuria syndrome suggest that the genetic defect leading to the syndrome might be located on chromosome 13.