Herein, we evaluated cervical samples from normal tissue or HPV-infected tissue, to determine if the relative nuclear/cytoplasmic ratio (NA/CA) and the presence of nonclassical cytological criteria are a novel cytological criterion for the diagnosis of HPV. Significantly, larger NA/CA ratios were found for the HPV−ATYPIA+ and HPV+ATYPIA+ groups compared with HPV−ATYPIA− group, regardless of collection method. For the samples collected with a spatula, only three samples from the HPV−ATIPIA− group showed four or more nonclassical parameters (i.e., were positive), while a larger number of the samples in the HPV−ATYPIA+, HPV+ATYPIA−, and HPV+ATYPIA+ groups were positive (13, 4, and 13 samples, respectively). Among those collected with a brush, no sample showed four or more nonclassical criteria in the HPV−ATYPIA− group, while a number of samples were positive in the HPV−ATYPIA+, HPV+ATYPIA−, and HPV+ATYPIA+ groups (4, 3, and 4 samples, respectively). HPV infection was associated with significant morphometrical changes; no increase in the NA/CA ratio was found in the HPV+ATYPIA- samples, compared with the HPV−ATIPIA− samples collected with either a spatula or a brush. In conclusion, by including nonclassical cytological criteria into the patient diagnosis, we were able to reduce the number of false negative and false positive HPV diagnoses made using conventional cytology alone. Diagn. Cytopathol. 2013;41:785–792. © 2013 Wiley Periodicals, Inc.