Background: Interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal tract. By connecting neuronal cells and smooth muscle cells, ICCs provide a continuous network responsible for the spontaneous electrical and mechanical activity of smooth muscle cells. In this study we tested the hypothesis that the absent peristalsis and impaired relaxation of the lower esophageal sphincter in achalasia are due to disappearance of ICCs in the muscular layer (muscularis externa or propria) of the esophagus.Methods: Representative distal esophageal tissue specimens from nine patients who underwent esophagectomy for end stage achalasia were identified from the medical records. The diagnosis of achalasia was confirmed in all patients by manometry. Normal distal esophageal tissue specimens from ten patients who underwent esophagectomy for squamous cell carcinoma of the mid- or upper esophagus were used for controls. Formalin-fixed, paraffin-embedded tissues were sectioned and stained for CD117/c-kit using standard antigen retrieval immunohistochemical techniques. CD117-positive ICCs were distinguished from CD117-positive mast cells on the basis of their stellate morphology and by the use of CD117/mast cell tryptase double immunostainining. ICCs located in the circular and longitudinal layers of the muscularis externa were counted in ten high power (x400) fields by two independent observers. Electron microscopy was performed to examine ICC ultrastructural features in achalasia.Results: ICCs were identified in the muscularis externa layer in all nine achalasia specimens and all 10 normal control specimens. The median and (range) ICC numbers per high power field in the achalasia specimens were 13.85 (7.0 - 23.9) for the inner circular muscle layer and 9.6 (7.8 - 11.5) for the outer longitudinal muscle layer, and 16.05 (6.5 - 26.7) and 10.9 (5.2 - 18.5) for the normal esophagus inner and outer muscluaris externa layers. The ICC density was not significantly different in achalasia compared to normal control specimens in any layer. Electron microscopy revealed the presence of cells with typical ICC ultrastructural appearance in achalasia tissue specimens. Conclusion: This study did not find a significant difference in the density of ICCs in achalasia compared to normal esophagus specimens. These results suggest that the motility defects that characterize achalasia result from insufficient neuronal input and perhaps impaired ICC activity rather than insufficient numbers of ICCs.