A sensitive and accurate stable isotope dilution assay was developed for the measurement of pipecolic acid in body fluids using electron capture negative ion mass fragmentography. The method utilizes [2H11]pipecolic acid as the internal standard. Sample preparation consisted of derivatization in aqueous solution (pH 11.5) of the amine moiety with methyl chloroformate to the N-methylcarbamate, followed by acidic ethyl acetate extraction (pH 2) and further derivatization of the carboxyl moiety to the pentafluorobenzyl ester. Normal values have been determined in cerebrospinal fluid (mean means = 0.041 mumol/l, range 0.010-0.120 mumol/l), in plasma of at term infants (age less than 1 wk, means = 5.73 mumol/l, range 3.75-10.8 mumol/l; age greater than 1 wk, means = 1.46 mumol/l, range 0.70-2.46 mumol/l), in urine of at term infants (age less than 6 mth, means = 32.5 mumol/g. creat., range 9.81-84.5 mumol/g. creat; age greater than 6 mth, means = 6.35 mumol/g. creat., range 0.15-13.6 mumol/g. creat.) and in amniotic fluid (means = 4.65 mumol/l, range 2.24-8.40 mumol/l). The utility of the method was demonstrated for the pipecolic acid quantification in these biofluids of patients with peroxisomal disorders. As affected fetuses with infantile Refsum's disease and Zellweger syndrome showed no significant elevation of pipecolic acid in their surrounding amniotic fluids, the measurement of pipecolic acid in amniotic fluid seemed not to be useful for prenatal diagnosis in these disorders.