Enteric neurons responding to chemical challenge of the mucosa have been characterized in animal models mainly in the myenteric plexus. However, in humans, the existence of enteric neurons responding to chemical stimulation of the mucosa remains currently unknown. Therefore, the aim of our study was to identify and characterize human submucosal neurons activated by mucosal challenge with butyrate or hydrochloric acid. Segments of human colon were placed in a modified Ussing chamber and incubated on the mucosal side with butyric acid (20 mM, pH 6.5), sodium butyrate (20 mM, pH 7.5), hydrochloric acid (10 mM, pH 6.5) or culture medium (pH 7.5). After 90 min of culture, tissues were fixed and microdissected to obtain whole mount preparation of submucosa containing the Meissner's plexus. Neuron specific enolase (NSE), c-Fos, vasoactive intestinal peptide (VIP) and substance P (SP) were detected using immunohistochemical methods. Tetrodotoxin (TTX, 1 microM) was used to inhibit neuronal activity. After 90 min of culture, butyric acid induced a significant 5.6-fold increase in the proportion of c-Fos-immunoreactive neurons compared to control (19 +/- 4% versus 4 +/- 1%, respectively, p < 0.001). 41 +/- 5% of c-Fos-immunoreactive neurons were VIP-immunoreactive and 3 +/- 2% were SP-immunoreactive. Butyric acid did not modify the proportion of VIP-immunoreactive neurons. The increase in c-Fos-immunoreactive neurons induced by butyric acid was reproduced with hydrochloric acid at the same pH but not with sodium butyrate. Finally, preincubation of the tissue with TTX prevented the effect of butyric acid. In conclusion, our results demonstrate that acidic mucosal challenge induced the activation of a population of human submucosal neurons with a specific neurochemical coding.