This study scans the efficacy of p16 immunostain on gynecological cytologies for the detection of high-grade lesions in ASCUS cases. This is a retrospective study that evaluates p16 immunostaining using frozen material from 109 cases diagnosed as ASCUS and followed for an average of 16 months. The diagnosis of ASCUS was made using liquid-based cytology (Thin Prep, Hologic, Marlborough, MA). High-risk HPV using Hybrid Capture II was performed. The immunohistochemical evaluation of p16 immunoreactivity was done following Wentzensen criteria. In the series p16 immunoreactivity showed a sensitivity and negative predictive value for HSIL, of 82.3 and 94.5% with specificity and positive predictive value of 100%. The sensitivity was similar to that found using the HC-II, though the specificity was much higher. The use of p16 immunostain in cytologic specimens applying the Wentzensen criteria helps to focus the attention in positive cells evaluating the cell changes, helps to detect some cells suspicious or diagnostic of HSIL, and could help to reclassify the ASCUS cases. Diagn.Cytopathol. 2011;39:110–114. © 2010 Wiley-Liss, Inc.