The possible involvement of serotonin (5HT) in mediating the inhibition of episodic LH release produced by electrical stimulation of the arcuate nucleus (ARH) of ovariectomized rats was investigated. Animals were pretreated with p-chlorophenylalanine (PCPA) an inhibitor of 5HT synthesis, or PCPA and 5-hydroxytryptophan (5HTP). Unanesthetized, unrestrained rats were bled continuously through indwelling cannulae prior to the onset of stimulation, and bleeding continued for 3 h during which electrical stimulation was applied for one or two 60 min periods separated by a 60 min nonstimulation period. Whole blood was analyzed for LH by radioimmunoassay. Brain 5HT levels were determined in individual rats by a fluorometric method. PCPA caused a significant depletion in brain 5HT levels by 71h. Administration of 30 or 120 mg/kg 5HTP to PCPA treated rats resulted, respectively, either in a restoration of normal brain 5HT levels, or a 3 1/2-fold increase above controls. Although stimulation of the ARH inhibits episodic LH release in ovariectomized rats, following depletion of brain 5HT levels with PCPA stimulation of the ARH markedly increased LH release. This increase lasted for most if not all of the stimulation period and was followed by a 1 h period of little or no LH secretion. These increases were not seen during stimulation in other hypothalamic areas outside the ARH. Reestablishment of normal, or even further increasing, brain 5HT levels in PCPA treated rats with 5HTP greatly reduced the extent of the increase in LH release seen during ARH stimulation, but only restored inhibition of LH secretion in a few animals. However, a decrease in LH rt 1) a decrease in brain 5HT levels reverses the effect of ARH stimulation on LH release from inhibition to excitation, and 2) repletion of 5HT levels greatly reduces the magnitude of this increased LH release. These data suggest that 5HT may be involved in mediating the inhibition of episodic LH secretion by electrical stimulation of the ARH. The inability of 5HTP to restore completely inhibition of episodic LH release during ARH stimulation suggests that a substance other than 5-HT may also be involved in mediating this response.