Although increasing evidence suggests that anatomically defined subpopulations of serotonergic neurons have unique stress-related functional properties, the topographical distribution of the serotonergic neurons involved in responses to stress-related stimuli have not been well-defined. Inspiration of air containing elevated concentrations of carbon dioxide (CO(2); hypercarbic gas exposure) at high concentrations activates both hypothalamic-pituitary-adrenal axis and sympathetic responses in rats and humans. In order to determine the effects of acute hypercarbic gas exposure on subpopulations of topographically organized serotonergic neurons, conscious adult male rats were placed in flow cages and exposed to either atmospheric air or increasing environmental CO2 concentrations (from baseline concentrations up to 20% CO2) for 5min. The presence of immunoreactivity for the protein product of the immediate-early gene c-fos was used as a measure, at the single cell level, of functional cellular responses within subpopulations of serotonergic, noradrenergic and adrenergic neurons. Rats exposed to hypercarbic gas had increased numbers of c-Fos/tryptophan hydroxylase immunoreactive (ir) and c-Fos/tyrosine hydroxylase-ir neurons in specific topographically organized subdivisions of brainstem nuclei, compared to control rats. Within serotonergic cell groups (B1-B9), the most striking effects occurred in a subpopulation of large, multipolar serotonergic neurons within the ventrolateral periaqueductal grey and ventrolateral part of the dorsal raphe nucleus, a region implicated in serotonin-dependent suppression of stress-induced sympathetic outflow and serotonin-dependent inhibition of 'fight or flight' behaviour. These findings have important implications for understanding the role of serotonergic systems in the modulation of stress-related physiology and behaviour and stress-related neuropsychiatric disorders.