Abstract A high-performance liquid chromatographic method for the simultaneous determination of perhexiline and its major metabolites, the cis- and trans-monohydroxyperhexilines M 1 and M 3, respectively, in human plasma or urine has been developed. Perhexiline and its metabolites are extracted from plasma or urine and derivatized with 1-fluoro-2,4-dinitrobenzene. The extracted dinitrophenyl derivatives of drug and metabolites are separated on a Spherisorb S5 ODS column by gradient elution. The limits of detection for perhexiline and its monohydroxy metabolites were 15 and 3 ng/ml, respectively. The inter-assay coefficients of variation for 100 ng/ml perhexiline, 100 ng/ml M 1 and 400 ng/ml M 3 were 10.5, 7.6 and 5.6%, respectively ( n = 9). The method has been employed in a limited kinetic study with five healthy adult male volunteers who received 150-mg and 300-mg Pexid tablets at an interval of one week. In four subjects perhexiline exhibited marked first pass effects, with plasma M 1 levels higher than unchanged perhexiline; in the urine M 1 was the predominant metabolite except in one subject who had higher M 3 than M 1 in the 300-mg Pexid study. The fifth subject exhibited a defective capacity to hydroxylate perhexiline; M 1 and M 3 were not detectable in plasma, and the urinary excretion of the monohydroxyperhexilines was relatively less, with M 3 present in higher amounts than M 1.