Background: A decrease in ganglionic cells associated to a mononuclear cell infiltrate characterize the myenteric plexus of patients with esophageal achalasia. Although several studies failed to demonstrate any viral DNA within the esophageal myenteric plexus, an autoimmune process triggered by Herpes virus (HSV)-1 may be responsible for the ganglionic cell damage. Therefore this study was designed to test the reactivity of lymphocytes isolated from the myenteric plexus to HSV1 antigens. Methods: During laparoscopic myotomy, 15 mm long strips of esophageal muscle were harvested, whereas muscle strips obtained from transplant multi-organ donors were used as controls. Muscle strips were digested with collagenase to obtain a single cell suspension and then mononuclear cells were purified through a Percoll gradient. As a specific indicator of CD4+ activation in response to HSV1 antigens, lymphocytes (100000 per well) were cultured in medium alone or containing UV-inactivated HSV1 (strain 16 wild type, infection ratio 1:1.5). After an initial 48 hrs 3H-thymidine (1 micro Curie) was added, then radioactivity incorporation was determined after a further 24 hrs incubation. A second fragment of esophageal muscle was used to extract total tissue RNA and DNA. Tissue DNA was used as a template to run the PCR using oligonucleotide primer pairs specific for HSV1 thymidine kinase (TK). Results: 6 patients with naive achalasia (4 M - 2 F, median age 49, range 20-67 yrs and median duration of dysphagia 40, range 12-60 mos) and 3 normal controls were studied. Direct and nested PCR using specific primers for HSV-1 TK revealed no amplificate of the expected size in controls and achalasia patients. After incubation of esophageal mononuclear cells from achalasia patients, there was a 1.6+/-0.2-fold increase in 3H-thymidine incorporation following exposure to UV-inactivated HSV-1, whereas the esophageal mononuclear cells obtained from controls did not respond to HSV-1 antigens (1.03+/-0.02 increase) (p<0.05) Conclusions: This study supports the hypothesis that lymphocytes reactive to HSV-1 antigens are present in the esophageal myenteric plexus and we speculate that these cells may trigger the inflammatory process leading to the disappearance of esophageal ganglionic cells.