AIM: To clarify whether perimuscular connective tissue contains more lymphatic vessels than the shallower layers in human gallbladders.METHODS: Lymphatic vessels were stained immunohistochemically with monoclonal antibody D2-40,which is a specific marker of lymphatic endothelium, in representative sections of 12 normal human gallbladders obtained at the time of resection for colorectal carcinoma liver metastases. In individual gallbladder specimens,nine high-power (× 200) fields with the highest lymphatic vessel density (LVD), termed hot spots, were identified for each layer (mucosa, muscle layer, and perimuscular connective tissue). In individual hot spots,the LVD and relative lymphatic vessel area (LVA) were measured microscopically using a computer-aided image analysis system. The mean LVD and LVA values for the nine hot spots in each layer were used for statistical analyses.RESULTS: In the mucosa, muscle layer, and perimuscular connective tissue, the LVD was 16.1 ± 9.2,35.4 ± 15.7, and 65.5 ± 12.2, respectively, and the LVA was 0.4 ± 0.4, 2.1 ± 1.1, and 9.4 ± 2.6, respectively.Thus, both the LVD and LVA differed significantly (P ＜0.001 and P ＜ 0.001, respectively; Kruskal-Wallis test)among the individual layers of the wall of the gallbladder,with the highest LVD and LVA values in the perimuscular connective tissue. Most (98 of 108) of the hot spots within the perimuscular connective tissue were located within 500 μm of the lower border of the muscle layer.CONCLUSION: The perimuscular connective tissue contains more and larger lymphatic vessels than the shallower layers in the human gallbladder. This observation partly explains why the incidence of lymph node metastasis is high in T2 (tumor invading the perimuscular connective tissue) or more advanced gallbladder carcinoma.