BACKGROUND The authors noted a significant increase in the diagnosis of atypical squamous cells of undetermined significance (ASCUS) in premenopausal women using oral contraceptive pills (OCP) (9%) versus women not on OCP (4%) using ThinPrep Pap smears (P = 0.02). The purpose of this study was to correlate these morphologic changes with human papillomavirus (HPV) DNA detection by in situ hybridization and clinical follow-up. METHODS Eighty-four ThinPrep Pap smears diagnosed as ASCUS or ASCUS favor low-grade squamous intraepithelial lesions (LGSIL) in young women receiving OCP were studied by HPV in situ hybridization. This information was correlated with colposcopic follow-up and AutoCyte Pap smear results. The authors also studied 80 ThinPreps from young women with a Pap smear diagnosis of unequivocal LGSIL where there was a corresponding biopsy (positive controls) and 40 ThinPreps diagnosed as within normal limits and that rescreened as such (negative controls). RESULTS The detection rate of HPV DNA by in situ hybridization was 33% (28 of 84) in ASCUS cases versus 90% (72 of 80) for cases of unequivocal LGSIL. A blinded review of the 29 ASCUS Pap smears performed by AutoCyte showed that 66% (19 of 29) were diagnosed as within normal limits. The majority of the cervical biopsies in the women with ASCUS by ThinPrep Pap smears were negative for dysplasia (29 of 39 [74%]) whereas 71 of 80 biopsies (88%) showed dysplasia in women with Pap smears diagnosed as unequivocal LGSIL. The periodic acid-Schiff (PAS) stain demonstrated that many of the cells with halos in the ThinPrep Pap smears that mimicked LGSIL contained glycogen. CONCLUSIONS The ThinPrep induces changes that mimic LGSIL in young women on OCP. These artifacts may reflect pressure-induced alteration of glycogen. PAS analysis and, more directly, HPV testing by in situ hybridization can help differentiate this mimicking from actual LGSILs.