The consistent histologic distinction of HPV-related precursor lesions from nonspecific epithelial changes is hampered by the fact that the scoring of nuclear atypia is subjective and nuclear alterations or cytoplasmic halos may occur with both HPV and non-HPV-related changes. To determine the potential role of another parameter--bi- or multinucleated cells--in the diagnosis of HPV-related lesions, we analyzed a series of epithelial alterations for anisokaryosis, hyperchromasia, and cytoplasmic halos and correlated their presence with the maximum of binucleate cells per high-power field (hpf) and the detection of HPV DNA. A positive correlation was seen between the presence of both anisokaryosis and hyperchromasia and the number of binucleate cells/hpf (p = 0.011) and the presence of HPV nucleic acids by in situ hybridization (p = 0.005). Only one of 23 (5.3%) known HPV-positive lesions did not exhibit any binucleate cells. These findings indicate that, although binucleation may be associated with a spectrum of both HPV and non-HPV related changes, it is most conspicuous and virtually always present in association with HPV-positive low-grade precursor lesions. Thus, this parameter may be useful to confirm the presence when other parameters suggest the diagnosis of a low-grade squamous intraepithelial lesion.