The activity of 5-hydroxytryptaminergic neurons has been estimated from measurements of: concentrations of 5-hydroxyindoleacetic acid; the ratio of the concentrations of 5-hydroxyindoleacetic acid to 5-hydroxytryptamine; the rate of accumulation of 5-hydroxytryptophan following the administration of an aromatic L-amino acid decarboxylase inhibitor (e.g., NSD 1015); the rate of accumulation of 5-hydroxytryptamine, and the rate of decline of 5-hydroxyindoleacetic acid following the administration of a monoamine oxidase inhibitor (e.g., pargyline). The purpose of the present study was to compare these different methods under conditions of changing neuronal impulse traffic produced by electrical stimulation of 5-hydroxytryptaminergic neurons. Male rats anesthetized with chloral hydrate were killed following 0, 15, or 30 min of electrical stimulation of the dorsal raphe nucleus at a frequency of 0, 5, or 10 Hz. The concentrations of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophan in nucleus accumbens, amygdala, suprachiasmatic nucleus, and dorsomedial nucleus were measured using HPLC coupled to an electrochemical detector. In each brain region, stimulation elicited an increase in the concentration of 5-hydroxyindoleacetic acid and the 5-hydroxyindoleacetic acid/5-hydroxytryptamine concentration ratio in saline-treated animals and an increase in 5-hydroxytryptophan accumulation in NSD 1015-treated animals, but did not alter the concentration of 5-hydroxytryptamine or 5-hydroxyindoleacetic acid in pargyline-treated rats. The results o f this study indicate that although the first three methods serve as valid indices of 5-hydroxytryptaminergic neuronal activity, the pargyline-dependent techniques are not responsive to changes in the rate of 5-hydroxytryptamine nerve firing.