AIMS: As tumour specimens and biopsy specimens become smaller, recognition of anatomical structures relevant for staging is increasingly challenging. So far no marker is known that reliably discriminates between muscularis propria (MP) and muscularis mucosae (MM) of the gastrointestinal tract. Recently, smoothelin expression has been shown to differ in MP and MM of the urinary bladder. We aimed to analyse the expression of smoothelin in the gastrointestinal tract in MP and MM in order to define a novel diagnostic tool to identify MM bundles. METHODS AND RESULTS: The expression of smoothelin was analysed immunohistochemically in comparison with alpha-smooth muscle actin (alpha-SMA) in specimens from colon, stomach and oesophagus (n = 107). In contrast to alpha-SMA, which equally stained MM and MP, absent or significantly weaker smoothelin expression was found in MM was found, which was particularly valid in colon and gastric specimens. CONCLUSIONS: The combination of smoothelin and SMA represents a robust marker to discriminate MM from MP in the gastrointestinal tract.