BACKGROUND The sensitivity of Papanicolaou smears for detecting endocervical adenocarcinoma in situ (AIS) is very low. A comprehensive cytological analysis of endocervical AIS is necessary to increase diagnostic accuracy. METHODS The subjects were 74 patients with pathologically-diagnosed AIS. A total of 140 Papanicolaou smears were reviewed to calculate the sensitivity of the Papanicolaou smears for detecting AIS and the incidence of sampling/screening/diagnostic errors. The cytological review was performed by 6 cytotechnologists, and the final cytological diagnosis was obtained at the consensus meeting. We classified the cases into three differentiation types; typical type (well-differentiated AIS), polymorphic type (poorly differentiated AIS), and mixed typical and polymorphic type. Three cytological subtypes (endocervical, endometrioid and intestinal subtypes) of AIS were also analyzed. RESULTS The sensitivity of the original Papanicolaou smears for the detection of AIS was 44.6%, while that for the detection of AIS and adenocarcinoma was 63.5%. The diagnostic accuracy of AIS increased to 78.5% in the final diagnosis. The common characteristic features were microbiopsies/hyperchromatic crowded groups (HCG) (82.0%) and mitotic figures (72.2%). The appearance of single cells (2.8%) was rare, and all the cervical cytology smears showed no evidence of necrotic tumor diathesis. The most common AIS was the typical type (41 cases, 67.2%) among all cytologically-diagnosed AIS or adenocarcinoma cases (61 cases). Although mixed typical and polymorphic AIS existed in 17 cases (27.9%), pure polymorphic AIS was very rare (3 cases, 4.9%). The endocervical subtype was the most predominant subtype (67.2%), followed by a few mixed subtypes. The important diagnostic keys for AIS cytology are as follows: (1) The appearance of microbiopsies/HCG (single-cell pattern is rare), (2) mitotic figures in the microbiopsies/HCG, (3) a lack of necrotic tumor diathesis in cases with polymorphic AIS, and (4) recognition of typical cytological subtypes. CONCLUSIONS The relatively low diagnostic accuracy AIS was caused by the underestimation of microbiopsies/HCG and the overestimation of polymorphic components. The typical cytological features of AIS are the presence of microbiopsies/HCG with mitotic figures in the absence of necrotic tumor diathesis in specimens containing endocervical samples. The recognition of infrequent AIS subtypes (endometrioid and intestinal subtypes) is also important.