Evidence suggests that 5-hydroxytryptamine 1A (5-HT(1A)) receptor-mediated autoregulation of serotonergic neuronal firing rates is impaired in stress-related neuropsychiatric disorders. In vitro models may provide insight into neural mechanisms underlying regulation of serotonergic systems. However, serotonin synthesis and tonic autoregulation of serotonergic neuronal firing rates are impaired in in vitro preparations lacking tryptophan. We describe the effects of perfusion of living rat brain slices with tryptophan on both 1) tissue concentrations of serotonin metabolites and 2) neuronal firing rates within the dorsal raphe nucleus. Brain slices were perfused with artificial cerebrospinal fluid lacking tryptophan for 4 h, followed by exposure to 1) 40 microM tryptophan (0-60 min) or 2) 0-400 microM tryptophan (23 min) and microdissected for analysis of indole concentrations. Parallel studies examined effects of tryptophan on neuronal firing rates and interactions with drugs expected to alter synaptic concentrations of serotonin. Tryptophan resulted in time-dependent and concentration-dependent increases in serotonin and serotonin metabolites, effects that were correlated with restoration of tonic autoinhibition of dorsal raphe nucleus neuronal firing rates. Inhibition of serotonin synthesis resulted in time-dependent and concentration-dependent increases in 5-hydroxtryptophan that correlated with reversal of the tryptophan-mediated autoinhibition of neuronal firing rates. Tryptophan modulated effects of several drugs on neuronal firing rates, including a selective 5-HT(1A) receptor antagonist (WAY-100635), a monoamine oxidase inhibitor (pargyline), a selective serotonin reuptake inhibitor (fluoxetine), and a serotonin-releasing agent (methylenedioxymethamphetamine). These studies support the hypothesis that tonic autoregulation of serotonergic neuronal firing rates is dependent on tryptophan availability and characterise conditions necessary to study this process in vitro.