p16 immunostaining has been examined to detect high-grade cervical intraepithelial neoplasia grade (CIN2+) in Pap cytology specimens. However, the utility of p16 in predicting CIN2+ in Pap specimens with atypical squamous cells of undetermined significance (ASC-US) or atypical squamous cells, cannot exclude high-grade squamous intraepithelial neoplasm (ASC-H), is controversial. In this study, we evaluated the utility of p16 immunostaining for predicting CIN2+ in 78 Pap specimens with ASC-US/ASC-H and compared the results in high-risk HPV DNA and the follow-up biopsies. p16 immunostaining was positive in 47% (37/78) of the Pap specimens. Of the 13 Pap specimens with follow-up biopsy results of CIN2+, 7 (54%) were positive for p16. p16 positivity in the Pap specimens was not significantly associated with a CIN2+ biopsy result (P = 0.76). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of p16 immunostaining for predicting CIN2+ were 54%, 52%, 19%, and 85%, respectively. High-risk HPV DNA was detected in 40% (31/78) of the Pap specimens. The sensitivity, specificity, PPV, and NPV of HPV DNA for predicting CIN2+ were 100%, 72%, 42%, and 100%, respectively. High-risk HPV genotypes were detected in six p16-negative specimens with follow-up biopsy results of CIN2+. Our findings suggest that the utility of p16 immunostaining for predicting CIN2+ in Pap specimens with ASC-US/ASC-H is limited. Scant abnormal cells in Pap specimens with ASC-US/ASC-H may have contributed to the low p16 sensitivity. Diagn. Cytopathol., 2011. © 2010 Wiley-Liss, Inc.