Hereditary fructose intolerance (HFI) is a potentially life-threatening disorder and can be suspected from a detailed nutritional history. The usefulness of 2 diagnostic procedures, fructose tolerance test (FTT) and aldolase assay on biopsied liver, was studied. A standardized intravenous FTT with 200 mg/kg b.w. was done on 11 children with HFI, 17 age-matched contrast children, 6 adults with HFI and 6 adult controls. Blood glucose, phosphorus, urate, magnesium and fructose were followed for 2 hours. By the FTT, each HFI individual was reliably distinguished from controls and contrasts and even from those with acute liver disease other than HFI. Both children with non-HFI hepatopathy examined by both procedures had a normal FTT in spite of reduced liver fructaldolase activity. HFI children responded to the FTT by earlier and more pronounced hypoglycemia than adults, and one girl converted to an adult type response between the ages 12 and 181/2 years. Responses of two HFI sibling pairs and of one set of monozygotic twins were typical for age, but resemblance was no greater than within the unrelated HFI probands. The intravenous FTT is judged a reliable diagnostic tool, simple and harmless if done in hospital. Essential fructosuria is readily diagnosed by the FTT, but fructose-1,6-diphosphatase deficiency and HFI are not differentiated with certainty. Liver biopsies were obtained from 35 children with HFI, 14 contrast persons and 10 controls (of which 9 organ donors) and examined enzymatically. Deficiency of fructaldolase was observed in all HFI children but also in some contrast children suffering from acute liver disease other than HFI. In these, HFI could only be excluded when the reduced activity of reference enzymes such as fructose-1,6-diphosphatase and glucose-6-phosphatase and liver histology were included in the evaluation. In one deceased HFI infant, fructaldolase was deficient in both, liver and kidney cortex. Extent of antibody activation and of heat inactivation of residual fructaldolase varied between unrelated HFI patients but not within families. These results did not contribute to diagnosis but further documented genetic heterogeneity of HFI. For diagnosis of HFI we recommend 1. immediate elimination of fructose from the diet, 2. the intravenous FTT after several weeks of fructose withdrawal, and 3., should diagnosis still be uncertain, laparoscopic liver biopsy for assay of fructaldose and of reference enzymes and for histology.