We describe the determination of the 4-O-methylated catecholamine metabolites 4-methoxy-3-hydroxyphenylacetic acid (iso-HVA), 4-methoxy-3-hydroxyphenylmandelic acid (iso-VMA) and 4-methoxy-3-hydroxyphenylethylene glycol (iso-MOPEG) in urine with use of mass fragmentography and deuterated internal standards. Normal values, expressed in terms of creatinine, are given as a function of age. Urinary excretion values during neuroblastoma, pheochromocytoma, and parkinsonism treated with L-DOPA, and during the intravenous administration of dopamine are discussed. For normal persons, the mean percentages (+/- SD) of 4-O-methylated metabolites relative to their 3-O-methylated analogs were 6.29 +/- 2.79% (iso-HVA/HVA), 0.70 +/- 0.57% (iso-VMA/VMA), and 0.77 +/- 0.53% (iso-MOPEG/MOPEG). We observed no significant changes in these parameters for the patients studied and conclude that the determination of 4-O-methylated catecholamine metabolites for these patients does not add information to the results obtained from their quantitatively more important 3-O-methylated analogs.