To determine why a vaginal self-collection tested for high-risk human papillomavirus (HR-HPV) by Hybrid Capture 2(R) (hc2) has lower sensitivity and specificity for cervical intraepithelial neoplasia Grade 2 or worse (> or = CIN 2), we collected 5 specimens (endocervix, upper and lower vagina, perineum, vaginal self-collection) from 2,625 women. Endocervical and self-collected specimens had HR-HPV tests by hc2. All 5 anogenital specimens were tested for 37 HPV genotypes [Linear Array(R), (LA)] from 397 women hc2 positive in endocervical or self-collected specimens and for a randomly selected 71 of 2,228 women hc2 negative on both specimens. Three hundred nintey-five women who screened positive by hc2 or had abnormal cytology underwent colposcopic evaluation. Of 47 women with > or = CIN 2, hc2 was positive in 97.9% (46/47) of endocervical and 80.9% (38/47), p = 0.008 of self-collected specimens. Seven of 9 women with > or = CIN 2 and negative self-collected hc2 tests were positive for HR-HPV by LA. Of 2,578 women without > or = CIN 2, hc2 was positive in 9.8% (253/2,578) of endocervical and 11.4% (294/2,578), p = 0.001 of self-collected specimens. Of the 41 more women without > or = CIN 2 that tested hc2 positive on the self-collected but negative on endocervical specimen, LA tested positive for HR-HPV in 24, negative for HPV in 11 and negative for HR-HPV but positive for low-risk HPV in 6. Lower sensitivity of self-collected specimens is secondary to lower levels of vaginal HR-HPV. The principal cause of the lower specificity of self-collected specimens is HR-HPV present solely in the vagina, which is not associated with > or = CIN 2.