The frequency of possible reasons for “atypical squamous cells” (ASC) overdiagnosis on Papanicolaou (Pap) smears was analyzed. Pap smears of 199 women with negative biopsy outcome after an ASC diagnosis were reviewed. Special attention was paid to presence of reproductive tract infections (RTIs), perimenopausal cells (PM cells), immature metaplastic cells, hormone-related alterations, and drying artefacts. Comparisons were made using χ2 test between the two ASC qualifiers and also between premenopausal and peri/postmenopausal women. Possible reasons for ASC overdiagnosis could be assigned on Pap smear review in 88/199 (44.2%) negative biopsies. Overall, PM cells were the most frequent reason for ASC overdiagnosis, being present in 35/199 (17.6%) smears. RTIs were the next most common cause (14.6%). PM cells were the most significant confounding factors for persistent ASC undetermined significance (ASC-US) over interpretation (20.2%) while in none of the cases these were interpreted as ASC-H (P = 0.004). Of these, 32 smears belonged to peri/postmenopausal women while only three to premenopausal women (P < 0.001). Immature metaplastic cells were significantly more frequent cause of ASC-H rather than ASC-US interpretation (P = 0.007). RTIs and drying artefacts were more frequently overcalled as ASC-US (in premenopausal women) while hormonal changes were interpreted as ASC-H. Hormone related changes, immature metaplastic cells and drying artefacts more commonly resulted in ASC interpretation in peri/ postmenopausal smears. The results of this study suggest that diligent screening can substantially reduce ASC overdiagnosis, thereby reducing the referrals/ follow ups. Diagn. Cytopathol. 2012. © 2011 Wiley Periodicals, Inc.