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The purpose of the present study was to clarify the spreading status of neoplastic cells in the cervical glands and surface epithelia in cervical intra-epithelial neoplasia (CIN) and microinvasive squamous cell carcinoma (MiSCC), and to evaluate the diagnostic usefulness of Ki-67 immunostaining from the gland involvement (GI) site. Cervical conization samples from 120 patients, including 110 with CIN (CIN1, n = 2; CIN2, n = 21; CIN3, n = 87) and 10 patients with MiSCC, was examined using HE and Ki-67 immunostaining. The linear extent, lateral extent in the surface epithelia and depth of GI were significantly increased from CIN1 to MiSCC. A significant correlation was found between the linear extent and lateral extent, between the linear extent and depth, and between the lateral extent and depth. These results indicated that the size of the surface epithelial lesion and the depth in CIN gradually increased in accordance with the grade of CIN, and that GI became deeper according to the increase in the size of the surface epithelial lesion. The Ki-67 labeling index in the GI site gradually increased from CIN1 to MiSCC, which indicated that Ki-67 immunostaining is a useful marker for the pathological diagnosis of CIN from the GI site.