Clinically, it is important to distinguish cervical intraepithelial neoplasia grade 3 (CIN3) lesions with and without coexisting adenocarcinoma in situ (AIS), but endocervical curetting can be false negative. The frequency of high-risk human papillomavirus genotypes in CIN3 patients with and without AIS differs. CIN3 epithelial cell cycle regulator expression may reflect these differences and thereby indicate coexistent AIS. G1 pathway epithelial cell cycle regulators (pRb, p53, cyclin D, p16) and Ki-67 were analyzed by quantitative immunohistochemistry in CIN3s with and without AIS. Compared with the normal cervical squamous epithelium, the CIN3 epithelium in small punch biopsies showed strong diffuse p16 and Ki-67 expression. CIN3s with coexistent AIS had a significantly lower percentage of pRb (P = .03)- and p53 (P = .03)-positive nuclei in the lower half of the epithelium than CIN3s without coexistent AIS. None of the 10 cases with values of either pRb-positive nuclei 30% or greater or p53-positive nuclei 15% or greater in the lower half of the epithelium had coexistent AIS, contrasting 8 (24%) of the 33 cases with both low values of p53- and pRb-positive nuclei. Combined low p53 and pRb expression in the lower half of the epithelium in punch biopsies is associated with coexistent AIS in the cone. Despite the fact that the results of the current study are interesting and potentially clinically relevant, it should be emphasized that they must be confirmed according to Good Laboratory Practice in independent patient groups, preferably also in a prospective study.