BACKGROUND Although low-grade squamous intraepithelial lesions (LSIL) most often are the result of infection by human papillomaviruses (HPV), a small proportion of women with LSIL have negative HPV tests. Using the Atypical Squamous Cells of Undetermined Significance/LSIL Triage Study (ALTS) population, the authors evaluated the significance of HPV-negative LSIL. METHODS Women with cytologic interpretations of LSIL by referral Papanicolaou (Pap) tests or enrollment ThinPrep tests (range, 1195-1476 women, depending on the specimen type and the reviewer) had HPV testing performed by both Hybrid Capture 2 and polymerase chain reaction (PCR)-based linear array for 27 HPV types. RESULTS Using 4 independent cytologic definitions of LSIL, only 3-11% of women with LSIL were found to have HPV-negative results on both HPV tests. The demographic characteristics of women with HPV-negative LSIL were consistent with those of a low-risk population; many were age > 35 years, and many reported no or only 1 recent sexual partner. The absolute risk of a histologic diagnosis of cervical intraepithelial neoplasia (CIN) Grade 3/carcinoma during the 2-year trial was lower for women with HPV-negative LSIL (range, 2-4%) compared with the absolute risks for oncogenic HPV-positive women with LSIL (range, 13-19%). However, at the next 6-month follow-up visit, 12%-32% of the women with HPV-negative LSIL had a positive HPV test. Finally, visual inspection of cervigrams demonstrated a clear association between a larger os and negative HPV test results compared with women who had HPV-positive LSIL. This may have influenced HPV sample adequacy. CONCLUSIONS Based on the ALTS data, the authors found no evidence to support the existence of HPV-negative LSIL as a distinct biologic entity related to the risk of cervical carcinoma. Such results appear to represent cytologic misinterpretations or falsely negative HPV tests.