To make reproducible diagnoses for oral carcinoma in situ (CIS), combined immunohistochemistry directed at the positioning of squamous cell proliferation (Ki-67) and differentiation (keratin (K) 13 and K19) was used, both of which support histological evaluations by providing biological evidence. Normal/hyperplastic epithelia was defined by K19+ cells only in the first basal layer, K13+ cells in the third basal and upper layers, and sporadic Ki-67+ cells in the second basal layer. These profiles indicated that a proliferating center of the oral epithelium is located in the parabasal cell layer, and K19 and K13 can be regarded as markers for basal and prickle cells, respectively. Epithelial dysplasia was characterized by irregular stratification of Ki-67+ cells and the absence of K19/K13 in proliferating cells. Irregular emerging of K19+ and K13+ cells in proliferating foci with unique stratification of atypical Ki-67+ cells indicated CIS. When the definition was applied, surgical margins in 172 recurrent cases were shown to contain CIS (39.4%) and squamous cell carcinoma (55.8%), indicating that the new diagnostic criteria for CIS reflected clinical behaviors of the cases. The results indicate that oral CIS contain more histological variations, especially those with definite keratinization, than what had been previously defined.