Glacial acetic acid (GAA) treatment minimizes the risk that bloody ThinPrep (Hologic, Marlborough, MA) Papanicolaou (Pap) test samples will be unsatisfactory for diagnosis. In our experience, GAA treatment also adversely affects the morphologic appearance of endocervical glandular cells. We analyzed a series of 92 consecutive GAA-treated Pap tests interpreted as atypical endocervical cells (AECs) and compared these with a control group of 130 samples with AECs in Pap tests that had not been treated with GAA to determine if GAA treatment increases the false-positive diagnosis of AECs. By using search data, the rates of AEC interpretations in the GAA-treated and GAA-untreated samples were calculated. Follow-up data, including human papillomavirus results and follow-up cytology and surgical pathology results, were collected. The GAA group had significantly fewer lesions on follow-up surgical pathology examinations than did the control group (6/69 [9%] vs 28/110 [25.5%]; P < or = .01). In our experience, GAA treatment increases the false-positive diagnosis of AECs.