The location, amount, and anatomic relationships of adenocarcinoma in situ (AIS) in 5 delayed second cone biopsy excision specimens and 21 definitive-therapy hysterectomy specimens were measured in relation to the neosquamocolumnar junction (nSCJ). All 5 biopsy specimens had 1 to 2 mm of AIS situated at the nSCJ. None had AIS in the proximal endocervix, despite positive or extremely close biopsy margins. Residual AIS in hysterectomy specimens was located proximal to the nSCJ in 19 (90%) of 21 cases. The mean distance between AIS and the nSCJ was 4.9 mm in 12 (86%) of 14 hysterectomy specimens. The mean maximum length of AIS was 4.6 mm in hysterectomy specimens and 1.1 mm in biopsy specimens. Some postbiopsy failures might be de novo neoplasms that begin at the nSCJ rather than recrudescence of persistent AIS. Small amounts of residual AIS following cone biopsy excision might be eradicated by the healing process. These 2 factors might underlie the disparate rates of residual AIS in hysterectomy specimens and postbiopsy excision failures and also explain the poor correlation between biopsy margin status and clinical failure. Factors that impact postbiopsy AIS eradication might be unrelated to de novo AIS beginning at the nSCJ.