Conventional endocervical adenocarcinoma in situ (cAIS) is typically strongly and diffusely positive for p16 with a high Ki67 index consistent with its frequent association with high-risk human papillomavirus (HPV) infection. The intestinal variant (iAIS) is less common, and its relationship to HPV infection has not been thoroughly examined. This study compares the clinicopathologic features, frequency of HPV infection, and expression of CDX2 and surrogate biomarkers of HPV infection (p16, Ki67) in cAIS with those of iAIS. A total of 86 cases with a diagnosis of AIS (49 iAIS, 37 cAIS) were identified from our multi-institutional files. Of these, 13 iAIS and 20 cAIS cases had slides and tissue available for histopathologic review, immunohistochemical analysis, and molecular tests. All 86 cases were used to evaluate clinical parameters; however, HPV DNA analysis and immunohistochemical analysis for p16, MIB-1, CDX2, and p53 were performed only on those cases with available slides or paraffin blocks. The average age at diagnosis was significantly higher in iAIS compared with that in cAIS (44.5 vs. 32.6 y) (P=0.0001). All 20 cAIS cases showed moderate to strong and diffuse p16 staining; however, only 9/13 iAIS cases showed this degree of p16 staining, whereas 4/13 (31%) iAIS cases showed weak and patchy distribution (P<0.02). Only 6/9 (67%) iAIS cases were positive for either HPV type 18 (5) or 33 (1), in contrast to 11/11 conventional cAIS (P=0.04). Similarly, 12/14 cAIS, but only 5/13 iAIS, cases showed a high Ki67 proliferative index. CDX2 was positive in all iAIS cases, whereas p53 was negative. Most iAIS cases are positive for high-risk HPV and show moderate to strong and diffuse p16 staining; however, a subset of iAIS shows variable staining with p16 and Ki67, is not associated with HPV, and occurs in a distinctly older age group suggesting an alternative pathogenesis. Awareness that iAIS can show variable staining for p16 and Ki67 is important when resolving problematic endocervical lesions, particularly in small biopsies with unusual p16 staining patterns.