A sensitive and accurate isotope dilution assay using electron capture negative ion mass fragmentography was developed for succinylacetone in amniotic fluid, plasma and urine. The method utilizes (D4)-5(3)-methyl-3(5)-isoxasole propionic acid as internal standard. Sample pretreatment consisted of oximation at pH less than 2 to 5(3)-methyl-3(5)-isoxasole propionic acid, clean up using liquid partition chromatography and further derivatization to the pentafluorobenzyl ester. Control values in plasma revealed a mean means = 0.044 mumol/l, range = 0.005-0.163 mumol/l, in urine means = 0.15 mumol/l, range 0.01-0.40 mumol/l corresponding to means = 0.03 mumol/mmol creat., range 0.01-0.14 mumol/mmol creat., and in amniotic fluid means = 0.016 mumol/l, range = 0.001-0.030 mumol/l. The utility of the method was demonstrated by quantification of succinylacetone in urine from patients with hereditary tyrosinemia type I (n = 8, excretion range 2.60-493.3 mumol/l corresponding to 0.67-197.3 mumol/mmol creat.) and in two amniotic fluid samples from fetuses affected with this disorder (concentration of succinylacetone 0.085 and 1.50 mumol/l, respectively). Maternal urine from a woman carrying an affected fetus did not show elevated urinary succinylacetone excretion.