Depth of invasion is one of the most important prognostic indicators in esophageal adenocarcinoma. Unlike other regions of the gastrointestinal tract, the esophagus in Barrett metaplasia frequently develops duplication of the muscularis mucosae (MM), but this feature is underrecognized, and its effect on appropriate staging of superficially invasive adenocarcinoma is unclear. We first randomly selected 50 esophageal resections for high-grade dysplasia or T1 adenocarcinoma in Barrett esophagus (BE) to evaluate the sensitivity and specificity of MM duplication for BE and its histologic characteristics, including percentage of the Barrett segment involved by MM duplication, origin of the duplicated muscle layer, and appearance of the tissue between duplicated MM. Twenty esophageal resections for squamous cell carcinoma served as controls. Next, to study the clinical significance of MM duplication, we evaluated 30 resections for BE that had superficial adenocarcinoma confined to regions of duplicated MM. Each case was classified as: depth of invasion (inner MM, space between duplicated MM, or outer MM), angiolymphatic invasion, and rate of lymph node metastasis. We observed MM duplication in 46 of 50 (92%) BE resections, involving 5% to >90% of the Barrett segment, in contrast to none in 20 (0%) resected squamous cell carcinoma, P<0.0001. In 5 (10%) cases, the MM was focally triplicated. The outer MM was continuous with the single MM beneath squamous epithelium, suggesting that outer MM represents the "original" muscle layer. The space between duplicated MM predominantly consisted of loose fibrovascular tissue similar to submucosa; in 15 (30%) cases, there were also areas of fibrosis or thin muscle strands joining the 2 MM layers. Of 30 adenocarcinomas invading duplicated MM, 10 (33%) invaded only inner MM, 12 (40%) invaded the space between MM, and 8 (27%) invaded the outer MM. Angiolymphatic invasion was present in 5 (17%) cases, and nodal metastases in 3 (10%, 1 case each of invasion into inner MM, between MM, and outer MM). These data show that MM duplication is a characteristic finding in BE, but it can pose difficulty in proper staging of superficial adenocarcinomas. The 17% rate of angiolymphatic invasion and 10% rate of lymph node metastases in our patients with invasion into duplicated MM suggest that these tumors can behave aggressively despite their technically intramucosal location.