Although a defect in catecholamine metabolism has been suggested in familial dysautonomia, the specific nature and extent of this abnormality has not been defined. Highly specific and sensitive assay techniques were used to measure the excretion of vanillylmandelic acid (VMA), 3-methoxy-4-hydroxyphenylethyleneglycol (HMPG), total metanephrines (TM), normetanephrine (NM), and homovanillic acid (HVA) of 52 dysautonomic patients and 180 normal subjects. Twenty parents of dysautonomic patients and age-matched control subjects were studied similarly. Patients with familial dysautonomia were found to excrete elevated quantities of HVA, diminished quantities of VMA and HMPG, and normal amounts of TM and NM in comparison with normal subjects. The excretion of the sum of the deaminated catecholamine metabolites proved to be more aberrant in familial dysautonomia than any other biochemical parameter measured. The findings of this study are more compatible with an abnormality in catecholamine synthesis rather than in release. Parents of dysautonomic subjects excreted normal amounts of vanillylmandelic acid and homovanillic acid.