Endocervical adenocarcinoma in situ (AIS) is believed to be a precursor of invasive disease; however, the biologic behavior of endocervical glandular "atypias" (GAs) is unclear. The purpose of this study was to evaluate the potential role of GA as a precursor of adenocarcinoma by assessment of the human papillomavirus (HPV) status of glandular lesions. We analyzed by polymerase chain reaction (PCR) 69 cases within a spectrum of endocervical glandular lesions encompassing invasive adenocarcinoma (IACA: 20 cases), AIS (21 cases), adenosquamous carcinoma (ASqCA: eight cases) and GA (20 cases) for HPV DNA sequences. Cervical adenocarcinoma is often associated with neoplastic squamous lesions (SL). In this study, after exclusion of AsqCA, 29 (47.5%) of 61 cases of endocervical glandular lesions were associated with an SL. The rate of HPV detection was not statistically different in adenocarcinoma with or without an SL (73.3% vs. 61.5%, respectively). In contrast, 64.3% of GAs with an SL (nine of 14 cases) were HPV positive, while only 16.7% of GAs without an SL (one of six cases) were positive. These findings suggest that HPV was preferentially associated with the concomitant SL rather than the GA. To localize the HPV sequences within the lesions, eight of the nine HPV-positive GAs with an SL were analyzed by in situ hybridization (ISH). Four cases were positive by ISH and showed hybridization of the probe only in the nuclei of squamous epithelial cells; in no lesion did the probe localize to the glandular epithelium. In our study, HPV Infection of the glandular epithelium of GAs unassociated with an SL appeared to be an uncommon event. None of the GAs were associated with low- or intermediate-risk HPV, and only the GA (which was high grade) unassociated with an SL contained a high-risk virus type. The possibility must be considered that pathogenetic mechanisms for squamous intraepithelial lesions may be different from those responsible for intraperitoneal glandular lesions.