BACKGROUNDIn a cytology‐based screening program intended to prevent anal cancer, the anal transformation zone (TZ) should be adequately sampled because it is the site most susceptible to the development of the cancer precursor, high‐grade squamous intraepithelial lesion (HSIL). An adequate TZ component is defined as comprising at least 10 rectal columnar or squamous metaplastic cells. In the current study, the authors examined whether the presence of a TZ component in anal cytology correlated with the detection of histological HSIL.METHODSIn a natural history study of anal human papillomavirus infection in homosexual men, all participants underwent liquid‐based cytology and high‐resolution anoscopy (HRA) with or without biopsy at each visit. True‐negative cytology (negative cytology with non‐HSIL biopsy or negative HRA), false‐negative cytology (negative cytology with HSIL biopsy), and true‐positive cytology (abnormal cytology with HSIL biopsy) were compared with regard to the presence or absence of a TZ component.RESULTSOf 617 participants, baseline results included 155 true‐positive results, 191 true‐negative results, and 31 false‐negative results. The absence of an adequate TZ component was found to be significantly higher for false‐negative (32.3%) than for either true‐positive (11.0%; P = .0034) or true‐negative (13.1%; P = .0089) results.CONCLUSIONSSignificantly more false‐negative cases lacked a TZ component compared with either true‐positive or true‐negative cases. TZ cells may be an important indicator of sample quality for anal cytology because, unlike cervical sampling, the anal canal is not visualized during cytology sampling. Cancer Cytopathol 2016;124:596‐601. © 2016 American Cancer Society.