Adeno-associated virus (AAV) is a ubiquitous human helper-dependent parvovirus which may interact with human papillomaviruses (HPV) to modify a woman's risk of cervical neoplasia. This analysis was nested in a cohort study of low-income women receiving Pap smears as part of their family planning services. We selected cases (55 with high-grade cervical squamous intraepithelial lesions (HSIL) and 162 with low-grade LSIL) and controls (96 women with normal cervical cytology) and analyzed cervical DNA for AAV, using PCR amplification/dot blot hybridization, and HPV, using hybrid capture I. AAV positivity was associated with a significantly reduced risk of HSIL (age and HPV-adjusted odds ratio (aOR) = 0.32) yet not with LSIL (aOR = 0.78); 53.8% of HSIL, 66.9% of LSIL, and 70.7% of controls were AAV+. AAV appears to interact with HPV to reduce SIL risk; relative to the HPV−/AAV+ exposure, the respective aORs for HSIL and HPV+/AAV−, HPV+/AAV+, and HPV−/AAV+ were 17.0, 6.9, and 3.5. AAV+ was not associated with age, race, HPV status, or sexual or reproductive risk factors. These results strongly suggest that AAV may play a protective or inhibitory role in late stage cervical carcinogenesis. This conclusion needs to be verified in additional epidemiologic studies.