1. The medullary raphe, within the ventromedial medulla (VMM), contains putative central respiratory chemoreceptors. To study the mechanisms of chemosensitivity in the raphe, rat VMM neurones were maintained in primary dissociated tissue culture, and studied using perforated patch-clamp recordings. Baseline electrophysiological properties were similar to raphe neurones in brain slices and in vivo. 2. Neurones were exposed to changes in CO2 from 5% to 3 or 9% while maintaining a constant [NaHCO3]. Fifty-one per cent of neurones (n = 210) did not change their firing rate by more than 20% in response to hypercapnic acidosis. However, 22% of neurones responded to 9% CO2 with an increase in firing rate ('stimulated'), and 27% of neurones responded with a decrease in firing rate ('inhibited'). 3. Chemosensitivity has often been considered an all-or-none property. Instead, a method was developed to quantify the degree of chemosensitivity. Stimulated neurones had a mean increase in firing rate to 298 +/- 215% of control when pH decreased from 7.40 to 7.19. Inhibited neurones had a mean increase in firing rate to 232 +/- 265% of control when pH increased from 7. 38 to 7.57. 4. Neurones were also exposed to isocapnic acidosis. All CO2-stimulated neurones tested (n = 15) were also stimulated by isocapnic acidosis, and all CO2-inhibited neurones tested (n = 19) were inhibited by isocapnic acidosis. Neurones with no response to hypercapnic acidosis also had no response to isocapnic acidosis (n = 12). Thus, the effects of CO2 on these neurones were mediated in part via changes in pH. 5. In stimulated neurones, acidosis induced a small increase in the after-hyperpolarization level of 1.38 +/- 1. 15 mV per -0.2 pH units, which was dependent on the level of tonic depolarizing current injection. In voltage clamp mode at a holding potential near resting potential, there were small and inconsistent changes in whole-cell conductance and holding current in both stimulated and inhibited neurones. These results suggest that pH modulates a conductance in stimulated neurones that is activated during repetitive firing, with a reversal potential close to resting potential. 6. The two subtypes of chemosensitive VMM neurones could be distinguished by characteristics other than their response to acidosis. Stimulated neurones had a large multipolar soma, whereas inhibited neurones had a small fusiform soma. Stimulated neurones were more likely than inhibited neurones to fire with the highly regular pattern typical of serotonergic raphe neurones in vivo. 7. Within the medullary raphe, chemosensitivity is a specialization of two distinct neuronal phenotypes. The response of these neurones to physiologically relevant changes in pH is of the magnitude that suggests that this chemosensitivity plays a functional role. Elucidating their mechanisms in vitro may help to define the cellular mechanisms of central chemoreception in vivo.