The origin of free 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG, a major metabolite of norepinephrine) in rat cerebrospinal fluid (CSF) was investigated using brain and spinal cord perfusions with artificial CSF, electrical stimulation of the locus coeruleus (LC) and the technique of retrograde cell labelling with horseradish peroxidase (HRP). The rates of appearance of MHPG into ventricular-cisternal and lumbar-cisternal perfusates were about 12.4 and 19.2 ng/hour respectively. Probenecid (200 mg/kg i.p.) did not alter the MHPG output in either preparation. Intravenous administration of 3 micrograms MHPG did not substantially enhance the outflow of the metabolite in the ventricular-cisternal perfusate, indicating that MHPG found in the perfusate is of central origin. Maximal activation of central norepinephrine (NE)-containing neurons of the LC by electrical stimulation induced a 50% increase of MHPG levels in the ventricular-cisternal perfusate. In addition HRP was found to be retrogradely transported from the lateral ventricle to these LC-neurons. These findings indicate that MHPG in CSF originates, at least in part from NE of nerve terminals adjacent to the cerebral ventricles, which have their origin in the LC. We calculated that the fraction of MHPG formed in the central nervous system, that was released into the CSF was about 34%. It is concluded that MHPG in CSF is a measure for changes in central NE turnover and that a considerable portion is dependent upon the activity of LC neurons.