Previous reports suggest that an important characteristic of chemosensitive neurons is an unusually large change of steady-state intracellular pH in response to a change in extracellular pH (pH_i/pH_o). To ask whether such a correlation exists between neurons from the medullary raphé (a chemosensitive brain region) and hippocampus (a non-chemosensitive region), we used BCECF to monitor pH_i in cultured neurons subjected to extracellular acid-base disturbances. In medullary raphé neurons, respiratory acidosis (5%9% CO₂) caused a rapid fall in pH_i(pH_i0.2) with no recovery and a large pH_i/pH_o = 0.71. Hippocampal neurons had a similar response, but with a slightly lower pH_i/pH_o (0.59). We further investigated a possible link between pH_i regulation and chemosensitivity by following the pH_i measurements on medullary raphé neurons with an immunocytochemical assay for tryptophan hydroxylase (a marker of serotonergic neurons). We found that the pH_i/pH_o of 0.69 for serotonergic neurons (which are stimulated by acidosis) was not different from either the pH_i/pH_o of 0.75 for non-serotonergic neurons (most of which are not chemosensitive), or from the pH_i/pH_o of hippocampal neurons. For both respiratory alkalosis (5%3% CO₂) and metabolic alkalosis (22 mM 35 mM HCO₃^-), pH_i/pH_o was 0.42 - 0.53 for all groups of neurons studied. The only marked difference between medullary raphé and hippocampal neurons was in response to metabolic acidosis (22 mM 14 mM HCO₃^-), which caused a large pH_i decrease in ~80% of medullary raphé neurons (pH_i/pH_o = 0.71), but relatively little pH_i decrease in 70% of the hippocampal neurons (pH_i/pH_o = 0.09). Our comparison of medullary-raphé and hippocampal neurons indicates that, except in response to metabolic acidosis, the neurons from the chemosensitive region do not have a uniquely high pH_i/pH_o. Moreover, regardless of whether neurons were cultured from the chemosensitive or the nonchemosensitive region, pH_i did not recover during any of the acid-base stresses.