The progression of cervical intraepithelial lesions to invasive cancer is associated with corresponding reductions in human papillomavirus (HPV) L1 capsid antigen (L1) expression. We sought to determine whether a similar loss of L1 occurs during anal carcinogenesis using immunohistochemistry on paraffin-embedded sections as well as INNO-LiPA HPV Genotyping (Innogenetics, Gent, Belgium) technology to determine HPV infection status. We analyzed 31 squamous cell carcinomas (SCCs), 26 SCCs in situ (SCC-IS), and 11 normal anal mucosae from 36 patients. High-risk HPV subtypes were detected in all patients. L1 nuclear staining was identified in 38% of SCC-IS; however, there was no detection in normal anal mucosae, SCC, or recurrent SCC. Of those SCC-IS associated with a concomitant invasive SCC, only 15% demonstrated nuclear L1 expression as compared to 62% of isolated SCC-IS (P = .02). Nuclear expression of L1 is lost in the progression of anal SCC-IS to SCC and may serve as a possible prognostic marker of enhanced malignant potential.