The treatment strategy for mesenchymal tumors of the gastrointestinal tract is based upon typing of the tumor. Especially differential diagnosis of gastrointestinal stromal tumors (GISTs) to leiomyomas is crucial for determining radicality of surgery. L1 cell adhesion molecule (CD171) plays an essential role in tumor progression. The aim of this study was to determine expression of L1 in GISTs, smooth muscle tumors, desmoid-type fibromatosis and peripheral nerve sheath tumors (PNSTs). We retrospectively analyzed a total of 129 surgically resected primary tumors or metastases of 72 GISTs, 29 smooth muscle tumors, seven PNSTs and 21 desmoid-type fibromatosis by immunohistochemistry for c-kit, CD34, smooth muscle actin, desmin, vimentin, S-100 and L1 expression. L1 expression was detected in 53 (74%) of 72 GISTs but in none of 29 smooth muscle tumors or 21 desmoid-type fibromatosis (P<0.01 by Fisher's test). In all, four (57%) of seven peripheral nerve sheath tumors were L1-positive. Survival analysis of 55 surgically completely resected GISTs presenting without metastasis at initial diagnosis revealed no tumor-specific death among L1-negative patients (P=0.13 by log-rank test; median follow-up time 41 months) and one recurrence was observed (P=0.12). Interestingly high levels of L1 were seen in tumor vascular endothelial cells of smooth muscle tumors and PNSTs, but not in GISTs. Our data show that L1 is highly expressed in GISTs but not in smooth muscle tumors and desmoid-type fibromatosis being important differential diagnoses. The trend towards a reduced survival of L1-positive patients in this study has to be further evaluated in future trials with higher patient numbers.