A simple, selective reverse-phase HPLC-fluorometric method is described for the determination of serotonin (5HT) in cisternal CSF of the rat. The mean (+/- SE) value of CSF 5HT observed in control adult rats was 457 +/- 83 pg/ml (N = 16). In an attempt to validate the measure as an index of extracellular, or functionally active, 5HT, groups of animals were treated with fenfluramine, amitriptyline, pargyline, pargyline plus tryptophan, and 5-hydroxytryptophan plus carbidopa. In all cases CSF 5HT appeared to reflect well the presumed effects of the agents on extracellular levels of 5HT. CSF 5HT was superior in this regard to brain 5HT, brain 5HIAA, or CSF 5HIAA levels. The measurement of cisternal CSF 5HT would appear to offer a convenient index of functionally active 5HT.